Engineered untranslated regions (utr) for aav production

ABSTRACT

The present disclosure describes methods and systems for use in the production of recombinant adeno-associated virus (rAAV) particles. In certain embodiments, the production process and system include engineered untranslated regions (UTR) which allow for the controlled expression of AAV capsid proteins, such as VP1, VP2 and VP3. In certain embodiments, the production process and system include engineered untranslated regions (UTR) which allow for the controlled expression of non-structural AAV replication proteins, such as Rep78 and Rep52.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/684,410, filed Jun. 13, 2018, entitled UNTRANSLATEDREGION (UTR) REGULATORY CONSTRUCTS, and U.S. Provisional PatentApplication No. 62/691,743, filed Jun. 29, 2018, entitled UNTRANSLATEDREGION (UTR) REGULATORY CONSTRUCTS, and U.S. Provisional PatentApplication No. 62/741,746, filed Oct. 5, 2018, entitled UNTRANSLATEDREGION (UTR) REGULATORY CONSTRUCTS FOR AAV CAP SEQUENCES, and U.S.Provisional Patent Application No. 62/741,779, filed Oct. 5, 2018,entitled UNTRANSLATED REGION (UTR) REGULATORY CONSTRUCTS FOR AAV REPSEQUENCES, the contents of which are each incorporated herein byreference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled20571511PCTSL.txt, created on Jun. 13, 2019, which is 30,920 bytes insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure describes methods and systems for use in theproduction of recombinant adeno-associated virus (rAAV) particles. Incertain embodiments, the production process and system includeengineered untranslated regions (UTR) which allow for the controlledexpression of AAV capsid proteins, such as VP1, VP2 and VP3. In certainembodiments, the production process and system include engineereduntranslated regions (UTR) which allow for the controlled expression ofnon-structural AAV replication proteins, such as Rep78 and Rep52.

BACKGROUND

AAVs have emerged as one of the most widely studied and utilized viralvectors for gene transfer to mammalian cells. See, e.g., Tratschin etal., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. GeneTher., 10(15):2445-2450 (1999), the contents of which are hereinincorporated by reference in their entirety. Adeno-associated viral(AAV) vectors are promising candidates for therapeutic gene delivery andhave proven safe and efficacious in clinical trials. The design andproduction of improved AAV particles for this purpose is an active fieldof study.

With the advent of development in the AAV field, there remains a needfor improved systems and methods for producing AAV vectors (such as AAVparticles) and corresponding gene therapy production materials such asBaculoviral Expression Vectors (BEVs).

SUMMARY

The present disclosure presents viral expression constructs whichinclude an expression control sequence and a protein-coding nucleotidesequence. In some embodiments, the viral expression construct includesan expression control sequence operably linked to a protein-codingnucleotide sequence. In some embodiments, the expression controlsequence includes an engineered polynucleotide. In some embodiments, theengineered polynucleotide is an engineered untranslated region (UTR). Insome embodiments, the engineered UTR polynucleotide functions as a 5′UTR.

In some embodiments, the protein-coding nucleotide sequence encodes astructural capsid protein. In some embodiments, the protein-codingnucleotide sequence encodes VP1, VP2, VP3 or a combination thereof. Insome embodiments, the protein-coding nucleotide sequence encodes anon-structural replication protein. In some embodiments, theprotein-coding nucleotide sequence encodes Rep78, Rep68, Rep52 and Rep40or a combination thereof.

In some embodiments, the expression control sequence includes apromoter. In some embodiments, the expression control sequence includesa late insect promoter or a very-later insect promoter. In someembodiments, the expression control sequence includes a promoterselected from Op-EI, EI, ΔEI, EI-1, pH, PIO, polH (polyhedron), ΔpolH,Dmhsp70, Hr1, Hsp70, 4×Hsp27 EcRE+minimal Hsp70, IE, IE-1, ΔIE-1, ΔIE,p10, Δp10, p5, p19, p35, p40, p6.9, and variations or derivativesthereof. In some embodiments, the expression control sequence includes apromoter selected from polH, p10, p6.9, and variations or derivativesthereof. In some embodiments, the expression control sequence includes ap10 promoter. In some embodiments, the expression control sequenceincludes a polH promoter.

In some embodiments, the engineered UTR includes a hairpin structure. Insome embodiments, the engineered UTR includes a hairpin structure of thepresent disclosure. In some embodiments, the hairpin structure includesa 5′ flanking region (i.e. upstream region), a stem region, a loopregion, and a 3′ flanking region (i.e. downstream region). In someembodiments, the hairpin structure includes a 5′ flanking region, a stemregion, a loop region, a stem-complement region, and a 3′ flankingregion.

In some embodiments, the hairpin structure is encoded by a hairpinsequence. In some embodiments, the hairpin sequence includes a leadersequence. In some embodiments, the 5′ flanking region (i.e. upstreamregion) is encoded by a 5′ flanking sequence. In some embodiments, the3′ flanking region (i.e. downstream region) is encoded by a 3′ flankingsequence. In some embodiments, the stem region is encoded by a stemsequence. In some embodiments, the loop region is encoded by a loopsequence. In some embodiments, the stem-complement region is encoded bya stem-complement sequence.

In some embodiments, an expression control sequence includes a startcodon for translation of a protein-coding nucleotide sequence. In someembodiments, the expression control sequence includes an engineered UTRwhich includes a start codon for translation of a protein-codingnucleotide sequence. In some embodiments, the engineered UTR includes ahairpin structure and a start codon for translation of a protein-codingnucleotide sequence. In some embodiments, the hairpin structure islocated 5′ of the start codon. In some embodiments, the start codon isincluded in the hairpin structure. In some embodiments, the start codonis included in the loop region of the hairpin structure. In someembodiments, the start codon is included in the stem-complement regionof the hairpin structure. In some embodiments, the start codon isincluded in the 3′ flanking (i.e. downstream) region of the hairpinstructure. In some embodiments, the distance between the 3′ end of theloop region and the start codon is from 1-30 nucleotides. In someembodiments, the distance between the 3′ end of the stem-complementregion and the start codon is from 1-30 nucleotides. In someembodiments, the start codon is ATG or ACG. In some embodiments, thestart codon is CTG, TTG, or GTG.

In some embodiments, the expression control sequence includes anengineered UTR which includes a Kozak nucleotide sequence or modifiedKozak nucleotide sequence of the present disclosure. In someembodiments, the Kozak nucleotide sequence or modified Kozak nucleotidesequence comprises an ATG start codon. In certain embodiments, themodified Kozak nucleotide sequence comprises an ACG start codon.

In some embodiments, the engineered 5′ UTR includes a hairpin structureselected from the hairpin structures presented in Table 1. In someembodiments, the engineered 5′ UTR includes a hairpin structure encodedby a hairpin nucleotide sequence selected from SEQ ID NO: 1-16. In someembodiments, the engineered 5′ UTR includes a hairpin structure encodedby a nucleotide sequence having at least 60% identity, at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to SEQ ID NO: 1-16.

In some embodiments, the engineered 5′ UTR includes a Hairpin 9structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a hairpin structure encoded by a hairpin nucleotidesequence comprising SEQ ID NO: 9. In some embodiments, the viralexpression constructs include an expression control sequence whichincludes a Hairpin 9 structure, and a protein-coding nucleotide sequencewhich encodes a structural capsid protein (such as VP1, VP2, VP3 or acombination thereof). In some embodiments, the viral expressionconstruct includes an expression control sequence which includes a p10promoter and a Hairpin 9 structure, and a protein-coding nucleotidesequence which encodes a structural capsid protein (such as VP1, VP2,VP3 or a combination thereof).

In some embodiments, the engineered 5′ UTR includes a Hairpin 12structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a hairpin structure encoded by a hairpin nucleotidesequence comprising SEQ ID NO: 12. In some embodiments, the viralexpression construct includes an expression control sequence whichincludes a Hairpin 12 structure, and a protein-coding nucleotidesequence which encodes a non-structural replication protein (such asRep78, Rep68, Rep52 and Rep40 or a combination thereof). In someembodiments, the viral expression constructs includes an expressioncontrol sequence which includes a polH promoter and a Hairpin 12structure, and a protein-coding nucleotide sequence which encodes anon-structural replication protein (such as Rep78, Rep68, Rep52 andRep40 or a combination thereof).

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a 5′ flanking region (i.e. upstream region) encoded by anucleotide sequence selected from atacgact, atac, and SEQ ID NO: 17-20.In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes an upstream region encoded by a nucleotide sequencehaving at least 60% identity, at least 65% identity, at least 70%identity, at least 75% identity, at least 80/o identity, at least 85%identity, at least 90% identity or at least 95% identity to a nucleotidesequence selected from atacgact, atac, and SEQ ID NO: 17-20.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a 3′ flanking region (i.e. downstream region) encoded bya nucleotide sequence selected from tttatggct, attatggct, tatatggct,ttaatggct, taaatggct, ataatggct, and SEQ ID NO: 34-35. In someembodiments, the engineered 5′ UTR includes a hairpin structure whichincludes an downstream region encoded by a nucleotide sequence having atleast 60% identity, at least 65% identity, at least 70% identity, atleast 75% identity, at least 80% identity, at least 85% identity, atleast 90% identity or at least 95% identity to a nucleotide sequenceselected from tttatggct, attatggct, tatatggct, ttaatggct, taaatggct,ataatggct, and SEQ ID NO: 34-35.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a stem region encoded by a nucleotide sequence selectedfrom cggc, ctgcc and SEQ ID NO: 21-31. In some embodiments, theengineered 5′ UTR includes a hairpin structure which includes an stemregion encoded by a nucleotide sequence having at least 60% identity, atleast 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity or atleast 95% identity to a nucleotide sequence selected from cggc, ctgccand SEQ ID NO: 21-31.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a stem complement region encoded by a nucleotide sequenceselected from ggcag and SEQ ID NO: 32-33. In some embodiments, theengineered 5′ UTR includes a hairpin structure which includes an stemcomplement region encoded by a nucleotide sequence having at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity or at least 95% identity to a nucleotide sequence selected fromggcag and SEQ ID NO: 32-33.

In some embodiments, the stem sequence and the stem-complement sequenceare 100% complementary (i.e. zero mismatches). In some embodiments, thestem sequence and the stem-complement sequence include zero, one, two,three, four or five mismatches. In some embodiments, the stem sequenceand the stem-complement sequence include one mismatch. In someembodiments, the stem sequence and the stem-complement sequence includetwo mismatches. In some embodiments, the stem sequence and thestem-complement sequence include three mismatches. In some embodiments,the stem sequence and the stem-complement sequence include 1-10mismatches.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a loop region encoded by a nucleotide sequence selectedfrom tttatggct and atctaa. In some embodiments, the engineered 5′ UTRincludes a hairpin structure which includes a loop region encoded by anucleotide sequence having at least 60% identity, at least 65% identity,at least 70% identity, at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity or at least 95% identity to anucleotide sequence selected from tttatggct and atctaa.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a leader sequence selected from SEQ ID NO: 36-51. In someembodiments, the engineered 5′ UTR includes a hairpin structure whichincludes a leader sequence selected from SEQ ID NO: 59-63. In someembodiments, the engineered 5′ UTR includes a hairpin structure whichincludes a leader sequence having at least 60% identity, at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from SEQ ID NO: 36-51. Insome embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a leader sequence having at least 60% identity, at least65% identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from SEQ ID NO: 59-63.

In some embodiments the length of the engineered 5′ UTR polynucleotideis from 20-200 nucleotides, from 20-100 nucleotides, from 20-50nucleotides or from 15 to 150 nucleotides.

In some embodiments, the engineered UTR improves the stability andprotein production capability of corresponding viral productionconstructs. In some embodiments, the engineered UTR modulates theexpression of one or more protein-coding nucleotide sequences. In someembodiments, the engineered UTR modulates ratios of proteins encoded bythe protein-coding nucleotide sequences. In some embodiments, theengineering of the UTR includes changing the strength, length, sequenceor tertiary structure of the UTR.

In some embodiments the engineered polynucleotide includes a start codon(e.g. ATG or ACG) and a 5′ UTR sequence which will down-regulate theexpression of a VP1 capsid protein relative to VP3. In some embodimentsthe engineered polynucleotide includes a start codon (e.g. ACG or ATG)and a 5′ UTR sequence which will up-regulate the expression of a VP1capsid protein relative to VP3.

In some embodiments the engineered polynucleotide includes a start codon(e.g. ATG or ACG) and a 5′ UTR sequence which will down-regulate theexpression of a VP2 capsid protein relative to VP3. In some embodimentsthe engineered polynucleotide includes a start codon (e.g. ACG or ATG)and a 5′ UTR sequence which will up-regulate the expression of a VP2capsid protein relative to VP3.

In some embodiments the engineered polynucleotide includes a start codon(e.g. ATG or ACG) and a 5′ UTR sequence which will down-regulate theexpression of a Rep78 capsid protein relative to Rep52. In someembodiments the engineered polynucleotide includes a start codon (e.g.ATG or ACG) and a 5′ UTR sequence which will up-regulate the expressionof a Rep78 capsid protein relative to Rep52.

In some embodiments the engineered polynucleotide includes a start codon(e.g. ATG or ACG) and a 5′ UTR sequence which will down-regulate theexpression of a Rep52 capsid protein relative to Rep78. In someembodiments the engineered polynucleotide includes a start codon (e.g.ATG or ACG) and a 5′ UTR sequence which will up-regulate the expressionof a Rep52 capsid protein relative to Rep78.

The present disclosure presents a method of altering the ratio ofstructural capsid proteins (i.e. VP proteins) expressed from aprotein-coding nucleotide sequence which encodes structural capsidproteins (such as VP1. VP2, VP3, or a combination thereof). In someembodiments, the method includes the use of an engineered polynucleotidewhich includes a start codon and a 5′ UTR sequence which willdown-regulate the expression of a VP1 and/or VP2 capsid proteinsrelative to VP3. In some embodiments, the method includes the use of anengineered polynucleotide which includes a start codon and a 5′ UTRsequence which will up-regulate the expression of a VP1 and/or VP2capsid proteins relative to VP3.

In some embodiments, the method results in a VP1:VP2:VP3 expressionratio of 0-3:0-3:10. In some embodiments, the method results in aVP1:VP2:VP3 expression ratio of 0.5-2: 0.5-2:10. In some embodiments,the method results in a VP1:VP2:VP3 expression ratio of about 1:1:10. Insome embodiments, the method results in a VP1:VP3 expression ratio of0-3:10, 0.5-2:10, or about 1:10. In some embodiments, the method resultsin a VP2:VP3 expression ratio of 0-3:10, 0.5-2:10, or about 1:10.

The present disclosure presents a method of altering the ratio ofnon-structural replication proteins (i.e. Rep proteins) expressed from aprotein-coding nucleotide sequence which encodes non-structuralreplications proteins (such as Rep78, Rep52, or a combination thereof).In some embodiments, the method includes the use of an engineeredpolynucleotide which includes a start codon and a 5′ UTR sequence whichwill down-regulate the expression of a Rep52 capsid proteins relative toRep78. In some embodiments, the method includes the use of an engineeredpolynucleotide which includes a start codon and a 5′ UTR sequence whichwill up-regulate the expression of a Rep52 capsid proteins relative toRep78. In some embodiments, the method includes the use of an engineeredpolynucleotide which includes a start codon and a 5′ UTR sequence whichwill down-regulate the expression of a Rep78 capsid proteins relative toRep52. In some embodiments, the method includes the use of an engineeredpolynucleotide which includes a start codon and a 5′ UTR sequence whichwill up-regulate the expression of a Rep78 capsid proteins relative toRep52.

In some embodiments, the method results in a ratio of p5 Rep proteins(Rep78 and Rep68) to p19 Rep proteins (Rep52 and Rep40) below 1:1. Insome embodiments, the method results in a p5:p19 ratio of 1:1-33,1:1-30, 1:1-25, 1:1-20, 1:1-15, 1:1-10, 1:1-6, 1:1-5, 1:1-4, 1:1-3 or1:1-2.

The details of various embodiments of the disclosure are set forth inthe description below. Other features, objects, and advantages of thedisclosure will be apparent from the description, drawings, and theclaims. In the description, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. In the case of conflict, the present descriptionwill control.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of thepresent disclosure, as illustrated in the accompanying figures. Thefigures are not necessarily to scale or comprehensive, with emphasisinstead being placed upon illustrating the principles of variousembodiments of the present disclosure.

FIG. 1 is a general illustration of one embodiment of an engineeredpolynucleotide (e.g., 5′ UTR) of the present disclosure.

FIG. 2A shows one embodiment of a viral expression construct of thepresent disclosure which includes a VP-coding region and an expressioncontrol sequence which includes a promoter and a hairpin structure.

FIG. 2B shows one embodiment of a viral expression construct of thepresent disclosure which include a Rep-coding region and an expressioncontrol sequence which includes a promoter and a hairpin structure.

FIG. 2C shows one embodiment of a viral expression construct of thepresent disclosure which includes both a Rep-coding region (withcorresponding hairpin structure and promoter) and a VP-coding region(with corresponding hairpin structure and promoter).

FIG. 3 is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with GC content in certain engineered UTRs of the presentdisclosure.

FIG. 4 is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with Weak Kozak sequences in certain engineered UTRs ofthe present disclosure. Weak Kozak sequences are underlined and ATGstart codon is in bold. FIG. 4 discloses SEQ ID NOS 64-66, respectively,in order of appearance.

FIG. 5 is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with certain engineered UTRs of the present disclosure.

FIG. 6A is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with HP (hairpin) 6, 7, 8, and 9 at 3, 4, and 5 days postinfection.

FIG. 6B is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with HP (hairpin) 10, 12, 13, 14, 15, and 16.

FIG. 7A is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with various Kozak sequences in certain engineered UTRs ofthe present disclosure.

FIG. 7B is a histogram of the Western blot analysis shown in FIG. 7A.

FIG. 8A is a Western blot analysis showing VP1, VP2 and VP3 expressioncorresponding with various Kozak sequences in certain engineered UTRs ofthe present disclosure.

FIG. 8B is a histogram of the Western blot analysis shown in FIG. 8A.

FIG. 8C shows the expression levels and production of VP1, VP2 and VP3for HP9-2D.

DETAILED DESCRIPTION I. Adeno-Associated Viruses (AAVS) Overview

Adeno-associated viruses (AAV) are small non-enveloped icosahedralcapsid viruses of the Parvoviridae family characterized by a singlestranded DNA viral genome. Parvoviridae family viruses consist of twosubfamilies: Parvovirinae, which infect vertebrates, and Densovirinae,which infect invertebrates. The Parvoviridae family includes theDependovirus genus which includes AAV, capable of replication invertebrate hosts including, but not limited to, human, primate, bovine,canine, equine, and ovine species.

The parvoviruses and other members of the Parvoviridae family aregenerally described in Kenneth I. Berns, “Parvoviridae: The Viruses andTheir Replication,” Chapter 69 in Fields Virology (3d Ed. 1996), thecontents of which are incorporated by reference in their entirety.

AAV have proven to be useful as a biological tool due to theirrelatively simple structure, their ability to infect a wide range ofcells (including quiescent and dividing cells) without integration intothe host genome and without replicating, and their relatively benignimmunogenic profile. The genome of the virus may be manipulated tocontain a minimum of components for the assembly of a functionalrecombinant virus, or viral particle, which is loaded with or engineeredto target a particular tissue and express or deliver a desired payload.

AAV Viral Genomes

The wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA)molecule approximately 5,000 nucleotides (nt) in length. Invertedterminal repeats (ITRs) traditionally cap the viral genome at both the5′ and the 3′ end, providing origins of replication for the viralgenome. While not wishing to be bound by theory, an AAV viral genometypically includes two ITR sequences. These ITRs have a characteristicT-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5′ and 3′ ends of the ssDNA which form anenergetically stable double stranded region. The double stranded hairpinstructures include multiple functions including, but not limited to,acting as an origin for DNA replication by functioning as primers forthe endogenous DNA polymerase complex of the host viral replicationcell.

The wild-type AAV viral genome further includes nucleotide sequences fortwo open reading frames, one for the four non-structural Rep proteins(Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the threecapsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genesor Cap genes). The Rep proteins are important for replication andpackaging, while the capsid proteins are assembled to create the proteinshell of the AAV, or AAV capsid. Alternative splicing and alternateinitiation codons and promoters result in the generation of fourdifferent Rep proteins from a single open reading frame and thegeneration of three capsid proteins from a single open reading frame.Though it varies by AAV serotype, as a non-limiting example, forAAV9/hu.14 (SEQ ID NO: 123 of U.S. Pat. No. 7,906,111, the contents ofwhich are herein incorporated by reference in their entirety) VP1 refersto amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refersto amino acids 203-736. In other words, VP1 is the full-length capsidsequence, while VP2 and VP3 are shorter components of the whole. As aresult, changes in the sequence in the VP3 region, are also changes toVP1 and VP2, however, the percent difference as compared to the parentsequence will be greatest for VP3 since it is the shortest sequence ofthe three. Though described here in relation to the amino acid sequence,the nucleic acid sequence encoding these proteins can be similarlydescribed. Together, the three capsid proteins assemble to create theAAV capsid protein. While not wishing to be bound by theory, the AAVcapsid protein typically includes a molar ratio of 1:1:10 ofVP1:VP2:VP3. As used herein, an “AAV serotype” is defined primarily bythe AAV capsid. In some instances, the ITRs are also specificallydescribed by the AAV serotype (e.g., AAV2/9).

For use as a biological tool, the wild-type AAV viral genome can bemodified to replace the rep/cap sequences with a nucleic acid sequenceincluding a payload region with at least one ITR region. Typically, inrecombinant AAV viral genomes there are two ITR regions. The rep/capsequences can be provided in trans during production to generate AAVparticles.

In addition to the encoded heterologous payload, AAV vectors may includethe viral genome, in whole or in part, of any naturally occurring and/orrecombinant AAV serotype nucleotide sequence or variant. AAV variantsmay have sequences of significant homology at the nucleic acid (genomeor capsid) and amino acid levels (capsids), to produce constructs whichare generally physical and functional equivalents, replicate by similarmechanisms, and assemble by similar mechanisms. See Chiorini et al., J.Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555-64 (1983);Chiorini et al., J. Vir. 73:1309-1319 (1999): Rutledge et al., J. Vir.72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), thecontents of each of which are incorporated herein by reference in theirentirety.

In certain embodiments, AAV particles, viral genomes and/or payloads ofthe present disclosure, and the methods of their use, may be asdescribed in WO2017189963, the contents of which are herein incorporatedby reference in their entirety.

AAV particles of the present disclosure may be formulated in any of thegene therapy formulations of the disclosure including any variations ofsuch formulations apparent to those skilled in the art. The reference to“AAV particles”. “AAV particle formulations” and “formulated AAVparticles” in the present application refers to the AAV particles whichmay be formulated and those which are formulated without limitingeither.

In certain embodiments, AAV particles of the present disclosure arerecombinant AAV (rAAV) viral particles which are replication defective,lacking sequences encoding functional Rep and Cap proteins within theirviral genome. These defective AAV particles may lack most or allparental coding sequences and essentially carry only one or two AAV ITRsequences and the nucleic acid of interest (i.e. payload) for deliveryto a cell, a tissue, an organ or an organism.

In certain embodiments, the viral genome of the AAV particles of thepresent disclosure includes at least one control element which providesfor the replication, transcription and translation of a coding sequenceencoded therein. Not all of the control elements need always be presentas long as the coding sequence is capable of being replicated,transcribed and/or translated in an appropriate host cell. Non-limitingexamples of expression control elements include sequences fortranscription initiation and/or termination, promoter and/or enhancersequences, efficient RNA processing signals such as splicing andpolyadenylation signals, sequences that stabilize cytoplasmic mRNA,sequences that enhance translation efficacy (e.g., Kozak consensussequence), sequences that enhance protein stability, and/or sequencesthat enhance protein processing and/or secretion.

According to the present disclosure, AAV particles for use intherapeutics and/or diagnostics include a virus that has been distilledor reduced to the minimum components necessary for transduction of anucleic acid payload or cargo of interest. In this manner, AAV particlesare engineered as vehicles for specific delivery while lacking thedeleterious replication and/or integration features found in wild-typeviruses.

AAV particles of the present disclosure may be produced recombinantlyand may be based on adeno-associated virus (AAV) parent or referencesequences. As used herein, a “vector” is any molecule or moiety whichtransports, transduces or otherwise acts as a carrier of a heterologousmolecule such as the nucleic acids described herein.

In addition to single stranded AAV viral genomes (e.g., ssAAVs), thepresent disclosure also provides for self-complementary AAV (scAAVs)viral genomes, scAAV vector genomes contain DNA strands which annealtogether to form double stranded DNA. By skipping second strandsynthesis, scAAVs allow for rapid expression in the cell.

In certain embodiments, the AAV viral genome of the present disclosureis a scAAV. In certain embodiments, the AAV viral genome of the presentdisclosure is a ssAAV.

Methods for producing and/or modifying AAV particles are disclosed inthe art, such as pseudotyped AAV particles (PCT Patent Publication Nos.WO200028004; WO200123001; WO2004112727: WO 2005005610 and WO 2005072364,the content of each of which is incorporated herein by reference in itsentirety).

AAV particles may be modified to enhance the efficiency of delivery.Such modified AAV particles can be packaged efficiently and be used tosuccessfully infect the target cells at high frequency and with minimaltoxicity. In certain embodiments the capsids of the AAV particles areengineered according to the methods described in US Publication NumberUS 20130195801, the contents of which are incorporated herein byreference in their entirety.

In certain embodiments, the AAV particles including a payload regionencoding a polypeptide or protein of the present disclosure, and may beintroduced into mammalian cells.

Inverted Terminal Repeats (ITRs)

The AAV particles of the present disclosure include a viral genome withat least one ITR region and a payload region. In certain embodiments,the viral genome has two ITRs. These two ITRs flank the payload regionat the 5′ and 3′ ends. The ITRs function as origins of replicationincluding recognition sites for replication. ITRs include sequenceregions which can be complementary and symmetrically arranged. ITRsincorporated into viral genomes of the present disclosure may beincluded of naturally occurring polynucleotide sequences orrecombinantly derived polynucleotide sequences.

The ITRs may be derived from the same serotype as the capsid, or aderivative thereof. The ITR may be of a different serotype than thecapsid. In certain embodiments, the AAV particle has more than one ITRIn a non-limiting example, the AAV particle has a viral genome includingtwo ITRs. In certain embodiments, the ITRs are of the same serotype asone another. In another embodiment, the ITRs are of different serotypes.Non-limiting examples include zero, one or both of the ITRs having thesame serotype as the capsid. In certain embodiments both ITRs of theviral genome of the AAV particle are AAV2 ITRs.

Independently, each ITR may be about 100 to about 150 nucleotides inlength. An ITR may be about 100-105 nucleotides in length, 106-110nucleotides in length, 111-115 nucleotides in length, 116-120nucleotides in length, 121-125 nucleotides in length, 126-130nucleotides in length, 131-135 nucleotides in length, 136-140nucleotides in length, 141-145 nucleotides in length or 146-150nucleotides in length. In certain embodiments, the ITRs are 140-142nucleotides in length. Non-limiting examples of ITR length are 102, 130,140, 141, 142, 145 nucleotides in length, and those having at least 95%identity thereto.

In certain embodiments, each ITR may be 141 nucleotides in length. Incertain embodiments, each ITR may be 130 nucleotides in length. Incertain embodiments, each ITR may be 119 nucleotides in length.

In certain embodiments, the AAV particles include two ITRs and one ITRis 141 nucleotides in length and the other ITR is 130 nucleotides inlength. In certain embodiments, the AAV particles include two ITRs andboth ITR are 141 nucleotides in length.

Promoters

In certain embodiments, the payload region of the viral genome includesat least one element to enhance the transgene target specificity andexpression (See e.g., Powell et al. Viral Expression Cassette Elementsto Enhance Transgene Target Specificity and Expression in Gene Therapy,2015: the contents of which are herein incorporated by reference in itsentirety). Non-limiting examples of elements to enhance the transgenetarget specificity and expression include promoters, endogenous miRNAs,post-transcriptional regulatory elements (PREs), polyadenylation (PolyA)signal sequences and upstream enhancers (USEs), CMV enhancers andintrons.

A person skilled in the art may recognize that expression of thepolypeptides of the present disclosure in a target cell may require aspecific promoter, including but not limited to, a promoter that isspecies specific, inducible, tissue-specific, or cell cycle-specific(see Parr et al., Nat. Med. 3:1145-9 (1997); the contents of which areherein incorporated by reference in their entirety).

In certain embodiments, the promoter is deemed to be efficient when itdrives expression of the polypeptide(s) encoded in the payload region ofthe viral genome of the AAV particle. In certain embodiments, thepromoter is a promoter deemed to be efficient when it drives expressionin the cell being targeted. In certain embodiments, the promoter is apromoter having a tropism for the cell being targeted. In certainembodiments, the promoter is a promoter having a tropism for a viralproduction cell.

In certain embodiments, the promoter drives expression of the payloadfor a period of time in targeted cells or tissues. Expression driven bya promoter may be for a period of 1-31 days (or any value or rangetherein), 1-23 months (or any value or range therein), 2-10 years (orany value or range therein), or more than 10 years. Expression may befor 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6years, 3-8 years, 4-8 years or 5-10 years. As a non-limiting example,the promoter can be a weak promoter for sustained expression of apayload in nervous (e.g. CNS) cells or tissues.

In certain embodiments, the promoter drives expression of thepolypeptides of the present disclosure for at least 1-11 months (or anyindividual value therein), 2-65 years (or any individual value therein),or more than 65 years.

Promoters may be naturally occurring or non-naturally occurring.Non-limiting examples of promoters include viral promoters, plantpromoters and mammalian promoters. In certain embodiments, the promotersmay be human promoters. In certain embodiments, the promoter may betruncated or mutated.

Promoters which drive or promote expression in most tissues include, butare not limited to, human elongation factor 1α-subunit (EF1α),cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chickenβ-actin (CBA) and its derivative CAG, β glucuronidase (GUSB), orubiquitin C (UBC). Tissue-specific expression elements can be used torestrict expression to certain cell types such as, but not limited to,muscle specific promoters, B cell promoters, monocyte promoters,leukocyte promoters, macrophage promoters, pancreatic acinar cellpromoters, endothelial cell promoters, lung tissue promoters, astrocytepromoters, or nervous system promoters which can be used to restrictexpression to neurons or subtypes of neurons, astrocytes, oroligodendrocytes.

Non-limiting examples of muscle-specific promoters include mammalianmuscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter,mammalian troponin 1 (TNNI2) promoter, and mammalian skeletalalpha-actin (ASKA) promoter (see, e.g. U.S. Patent Publication US20110212529, the contents of which are herein incorporated by referencein their entirety)

Non-limiting examples of tissue-specific expression elements for neuronsinclude neuron-specific enolase (NSE), platelet-derived growth factor(PDGF), platelet-derived growth factor B-chain (PDGF-β), synapsin (Syn),methyl-CpG binding protein 2 (MeCP2), Ca²⁺/calmodulin-dependent proteinkinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2),neurofilament light (NFL) or heavy (NFH), β-globin minigene nβ2,preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acidtransporter 2 (EAAT2) promoters. Non-limiting examples oftissue-specific expression elements for astrocytes include glialfibrillarv acidic protein (GFAP) and EAAT2 promoters. A non-limitingexample of a tissue-specific expression element for oligodendrocytesincludes the myelin basic protein (MBP) promoter.

In certain embodiments, the promoter may be less than 1 kb. The promotermay have a length of 200-800 nucleotides (or any value or rangetherein), or more than 800 nucleotides. The promoter may have a lengthbetween 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400,300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800,500-600, 500-700, 500-800, 600-700, 600-800 or 700-800.

In certain embodiments, the promoter may be a combination of two or morecomponents of the same or different starting or parental promoters suchas, but not limited to, CMV and CBA. Each component may have a length of200-800 nucleotides (or any value or range therein), or more than 800nucleotides. Each component may have a length between 200-300, 200400,200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700,300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800,600-700, 600-800 or 700-800. In certain embodiments, the promoter is acombination of a 382 nucleotide CMV-enhancer sequence and a 260nucleotide CBA-promoter sequence.

In certain embodiments, the viral genome includes a ubiquitous promoter.Non-limiting examples of ubiquitous promoters include CMV, CBA(including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC. GUSB (hGBp),and UCOE (promoter of HNRPA2B1-CBX3).

Yu et al. (Molecular Pain 2011, 7:63: the contents of which are hereinincorporated by reference in their entirety) evaluated the expression ofeGFP under the CAG, EF1α, PGK and UBC promoters in rat DRG cells andprimary DRG cells using lentiviral vectors and found that UBC showedweaker expression than the other 3 promoters and only 10-12% glialexpression was seen for all promoters. Soderblom et al. (E. Neuro 2015;the contents of which are herein incorporated by reference in itsentirety) evaluated the expression of eGFP in AAV8 with CMV and UBCpromoters and AAV2 with the CMV promoter after injection in the motorcortex. Intranasal administration of a plasmid containing a UBC or EF1αpromoter showed a sustained airway expression greater than theexpression with the CMV promoter (See e.g., Gill et al., Gene Therapy2001, Vol. 8, 1539-1546; the contents of which are herein incorporatedby reference in their entirety). Husain et al. (Gene Therapy 2009; thecontents of which are herein incorporated by reference in its entirety)evaluated an HOH construct with a hGUSB promoter, a HSV-1LAT promoterand an NSE promoter and found that the HOH construct showed weakerexpression than NSE in mouse brain. Passini and Wolfe (J. Virol. 2001,12382-12392, the contents of which are herein incorporated by referencein its entirety) evaluated the long term effects of the HβH vectorfollowing an intraventricular injection in neonatal mice and found thatthere was sustained expression for at least 1 year. Low expression inall brain regions was found by Xu et al. (Gene Therapy 2001, 8,1323-1332: the contents of which are herein incorporated by reference intheir entirety) when NFL and NFH promoters were used as compared to theCMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR. PPE, PPE+wpre, NSE (0.3 kb).NSE (1.8 kb) and NSE (1.8 kb+wpre). Xu et al. found that the promoteractivity in descending order was NSE (1.8 kb), EF, NSE (0.3 kb), GFAP.CMV, hENK, PPE. NFL and NFH. NFL is a 650-nucleotide promoter and NFH isa 920 nucleotide promoter which are both absent in the liver but NFH isabundant in the sensory proprioceptive neurons, brain and spinal cordand NFH is present in the heart. SCN8A is a 470 nucleotide promoterwhich expresses throughout the DRG, spinal cord and brain withparticularly high expression seen in the hippocampal neurons andcerebellar Purkinje cells, cortex, thalamus and hypothalamus (See e.g.,Drews et al. Identication of evolutionary conserved, functionalnoncoding elements in the promoter region of the sodium channel geneSCN8A, Mamm Genome (2007) 18:723-731; and Raymond et al. Expression ofAlternatively Spliced Sodium Channel α-subunit genes, Journal ofBiological Chemistry (2004) 279(44) 46234-46241; the contents of each ofwhich are herein incorporated by reference in their entireties).

Any of the promoters taught by the aforementioned Yu, Soderblom, Gill,Husain, Passini, Xu. Drews or Raymond may be used in the presentdisclosures.

In certain embodiments, the promoter is not cell specific.

In certain embodiments, the promoter is an ubiquitin c (UBC) promoter.The UBC promoter may have a size of 300-350 nucleotides. As anon-limiting example, the UBC promoter is 332 nucleotides. In certainembodiments, the promoter is a β-glucuronidase (GUSB) promoter. The GUSBpromoter may have a size of 350-400 nucleotides. As a non-limitingexample, the GUSB promoter is 378 nucleotides. In certain embodiments,the promoter is a neurofilament light (NFL) promoter. The NFL promotermay have a size of 600-700 nucleotides. As a non-limiting example, theNFL promoter is 650 nucleotides. In certain embodiments, the promoter isa neurofilament heavy (NFH) promoter. The NFH promoter may have a sizeof 900-950 nucleotides. As a non-limiting example, the NFH promoter is920 nucleotides. In certain embodiments, the promoter is a SCN8Apromoter. The SCN8A promoter may have a size of 450-500 nucleotides. Asa non-limiting example, the SCN8A promoter is 470 nucleotides.

In certain embodiments, the promoter is a frataxin (FXN) promoter. Incertain embodiments, the promoter is a phosphoglycerate kinase 1 (PGK)promoter. In certain embodiments, the promoter is a chicken β-actin(CBA) promoter, or variant thereof. In certain embodiments, the promoteris a CB6 promoter. In certain embodiments, the promoter is a minimal CBpromoter. In certain embodiments, the promoter is a cytomegalovirus(CMV) promoter. In certain embodiments, the promoter is a H1 promoter.In certain embodiments, the promoter is a CAG promoter. In certainembodiments, the promoter is a GFAP promoter. In certain embodiments,the promoter is a synapsin promoter. In certain embodiments, thepromoter is an engineered promoter. In certain embodiments, the promoteris a liver or a skeletal muscle promoter. Non-limiting examples of liverpromoters include human α-1-antitrypsin (hAAT) and thyroxine bindingglobulin (TBG). Non-limiting examples of skeletal muscle promotersinclude Desmin, MCK or synthetic C5-12. In certain embodiments, thepromoter is a RNA pol III promoter. As a non-limiting example, the RNApol III promoter is U6. As a non-limiting example, the RNA pol IIIpromoter is H1. In certain embodiments, the promoter is acardiomyocyte-specific promoter. Non-limiting examples ofcardiomyocyte-specific promoters include αMHC, cTnT, and CMV-MLC2k. Incertain embodiments, the viral genome includes two promoters. As anon-limiting example, the promoters are an EF1α promoter and a CMVpromoter.

In certain embodiments, the viral genome includes an enhancer element, apromoter and/or a 5′ UTR intron. The enhancer element, also referred toherein as an “enhancer,” may be, but is not limited to, a CMV enhancer,the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE,Synapsin, MeCP2, and GFAP promoter and the 5′ UTR/intron may be, but isnot limited to, SV40, and CBA-MVM. As a non-limiting example, theenhancer, promoter and/or intron used in combination may be: (1) CMVenhancer, CMV promoter, SV40 5′ UTR intron; (2) CMV enhancer, CBApromoter, SV 40 5′ UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM5′ UTR intron: (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter;(7) Synapsin promoter; (8) MeCP2 promoter and (9) GFAP promoter.

In certain embodiments, the viral genome includes an engineeredpromoter.

In another embodiment, the viral genome includes a promoter from anaturally expressed protein.

Untranslated Regions (UTRs)

By definition, wild type untranslated regions (UTRs) of a gene aretranscribed but not translated. Generally, the 5′ UTR starts at thetranscription start site and ends at the start codon and the 3′ UTRstarts immediately following the stop codon and continues until thetermination signal for transcription.

Features typically found in abundantly expressed genes of specifictarget organs may be engineered into UTRs to enhance the stability andprotein production. As a non-limiting example, a 5′ UTR from mRNAnormally expressed in the liver (e.g., albumin, serum amyloid A,Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, orFactor VIII) may be used in the viral genomes of the AAV particles ofthe present disclosure to enhance expression in hepatic cell lines orliver.

While not wishing to be bound by theory, wild-type 5′ untranslatedregions (UTRs) include features which play roles in translationinitiation. Kozak sequences, which are commonly known to be involved inthe process by which the ribosome initiates translation of many genes,are usually included in 5′ UTRs. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (ATG), which is followed by another‘G’. Incertain embodiments, the 5′ UTR in the viral genome includes a Kozaksequence. In certain embodiments, the 5′ UTR in the viral genome doesnot include a Kozak sequence.

While not wishing to be bound by theory, wild-type 3′ UTRs are known tohave stretches of Adenosines and Uridines embedded therein. These AUrich signatures are particularly prevalent in genes with high rates ofturnover. Based on their sequence features and functional properties,the AU rich elements (AREs) can be separated into three classes (Chen etal, 1995, the contents of which are herein incorporated by reference inits entirety): Class I AREs, such as, but not limited to, c-Myc andMyoD, contain several dispersed copies of an AUUUA motif within U-richregions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a,possess two or more overlapping UUAUU UA(U/A)(U/A) nonamers. Class IIIARES, such as, but not limited to, c-Jun and Myogenin, are less welldefined. These U rich regions do not contain an AUUUA motif. Mostproteins binding to the AREs are known to destabilize the messenger,whereas members of the ELAV family, most notably HuR, have beendocumented to increase the stability of mRNA. HuR binds to AREs of allthe three classes. Engineering the HuR specific binding sites into the3′ UTR of nucleic acid molecules will lead to HuR binding and thus,stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides. Whenengineering specific polynucleotides, (e.g., payload regions of viralgenomes), one or more copies of an ARE can be introduced to makepolynucleotides less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.

In certain embodiments, the 3′ UTR of the viral genome may include anoligo(dT) sequence for templated addition of a poly-A tail.

In certain embodiments, the viral genome may include at least one miRNAseed, binding site or full sequence. MicroRNAs (or miRNA or miR) are19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acidtargets and down-regulate gene expression either by reducing nucleicacid molecule stability or by inhibiting translation. A microRNAsequence includes a “seed” region, i.e., a sequence in the region ofpositions 2-8 of the mature microRNA, which sequence has perfectWatson-Crick complementarity to the miRNA target sequence of the nucleicacid.

In certain embodiments, the viral genome may be engineered to include,alter or remove at least one miRNA binding site, sequence or seedregion.

Any UTR from any gene known in the art may be incorporated into theviral genome of the AAV particle. These UTRs, or portions thereof, maybe placed in the same orientation as in the gene from which they wereselected, or they may be altered in orientation or location. In certainembodiments, the UTR used in the viral genome of the AAV particle may beinverted, shortened, lengthened, made with one or more other 5′ UTRs or3′ UTRs known in the art. As used herein, the term “altered” as itrelates to a UTR, means that the UTR has been changed in some way inrelation to a reference sequence. For example, a 3′ or 5′ UTR may bealtered relative to a wild type or native UTR by the change inorientation or location as taught above or may be altered by theinclusion of additional nucleotides, deletion of nucleotides, swappingor transposition of nucleotides.

In certain embodiments, the viral genome of the AAV particle includes atleast one artificial UTRs which is not a variant of a wild type UTR.

In certain embodiments, the viral genome of the AAV particle includesUTRs which have been selected from a family of transcripts whoseproteins share a common function, structure, feature or property.

Polyadenylation Sequence

In certain embodiments, the viral genome of the AAV particles of thepresent disclosure include at least one polyadenylation sequence. Theviral genome of the AAV particle may include a polyadenylation sequencebetween the 3′ end of the payload coding sequence and the 5′ end of the3′ ITR.

In certain embodiments, the polyadenylation sequence or “polyA sequence”may range from absent to about 500 nucleotides in length. Thepolyadenylation sequence may be, but is not limited to, 1-500nucleotides in length (or any value or range therein).

In certain embodiments, the polyadenylation sequence is 127 nucleotidesin length. In certain embodiments, the polyadenylation sequences is 477nucleotides in length. In certain embodiments, the polyadenylationsequence is 552 nucleotides in length.

Linkers

Viral genomes of the present disclosure may be engineered with one ormore spacer or linker regions to separate coding or non-coding regions.

In certain embodiments, the payload region of the AAV particle mayoptionally encode one or more linker sequences. In some cases, thelinker may be a peptide linker that may be used to connect thepolypeptides encoded by the payload region. Some peptide linkers may becleaved after expression to separate polypeptide domains, allowingassembly of mature protein fragments. Linker cleavage may be enzymatic.In some cases, linkers include an enzymatic cleavage site to facilitateintracellular or extracellular cleavage. Some payload regions encodelinkers that interrupt polypeptide synthesis during translation of thelinker sequence from an mRNA transcript. Such linkers may facilitate thetranslation of separate protein domains (e.g., heavy and light chainantibody domains) from a single transcript. In some cases, two or morelinkers are encoded by a payload region of the viral genome.

In certain embodiments, payload regions encode linkers including furincleavage sites. Furin is a calcium dependent serine endoprotease thatcleaves proteins just downstream of a basic amino acid target sequence(Arg-X-(Arg/Lys)-Arg) (Thomas, G., 2002. Nature Reviews Molecular CellBiology 3(10): 753-66; the contents of which are herein incorporated byreference in its entirety). Furin is enriched in the trans-golgi networkwhere it is involved in processing cellular precursor proteins. Furinalso plays a role in activating a number of pathogens. This activity canbe taken advantage of for expression of polypeptides of the disclosure.

In certain embodiments, payload regions encode linkers including 2Apeptides. 2A peptides are small “self-cleaving” peptides (18-22 aminoacids) derived from viruses such as foot-and-mouth disease virus (F2A),porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), or equinerhinitis A virus (E2A). The 2A designation refers specifically to aregion of picornavirus polyproteins that lead to a ribosomal skip at theglycyl-prolyl bond in the C-terminus of the 2A peptide (Kim, J. H. etal., 2011. PLoS One 6(4): e18556; the contents of which are hereinincorporated by reference in its entirety). This skip results in acleavage between the 2A peptide and its immediate downstream peptide. Asopposed to IRES linkers, 2A peptides generate stoichiometric expressionof proteins flanking the 2A peptide and their shorter length can beadvantageous in generating viral expression vectors.

In certain embodiments, payload regions encode linkers including IRES.Internal ribosomal entry site (IRES) is a nucleotide sequence (>500nucleotides) that allows for initiation of translation in the middle ofan mRNA sequence (Kim, J. H. et al., 2011. PLoS One 6(4): e18556: thecontents of which are herein incorporated by reference in its entirety).Use of an IRES sequence ensures co-expression of genes before and afterthe IRES, though the sequence following the IRES may be transcribed andtranslated at lower levels than the sequence preceding the IRESsequence.

In certain embodiments, the payload region may encode one or morelinkers including cathepsin, matrix metalloproteinases or legumaincleavage sites. Such linkers are described e.g. by Cizeau and Macdonaldin International Publication No. WO2008052322, the contents of which areherein incorporated in their entirety. Cathepsins are a family ofproteases with unique mechanisms to cleave specific proteins. CathepsinB is a cysteine protease and cathepsin D is an aspartyl protease. Matrixmetalloproteinases are a family of calcium-dependent and zinc-containingendopeptidases. Legumain is an enzyme catalyzing the hydrolysis of(-Asn-Xaa-) bonds of proteins and small molecule substrates.

In certain embodiments, payload regions may encode linkers that are notcleaved. Such linkers may include a simple amino acid sequence, such asa glycine rich sequence. In some cases, linkers may include flexiblepeptide linkers including glycine and serine residues. The linker mayinclude flexible peptide linkers of different lengths, e.g. nxG4S, (SEQID NO: 68) where n=1-10, and the length of the encoded linker variesbetween 5 and 50 amino acids. In a non-limiting example, the linker maybe 5×G4S (SEQ ID NO: 69). These flexible linkers are small and withoutside chains so they tend not to influence secondary protein structurewhile providing a flexible linker between antibody segments (George, R.A., et al., 2002. Protein Engineering 15(11): 871-9; Huston, J. S. etal., 1988. PNAS 85:5879-83; and Shan. D. et al., 1999. Journal ofImmunology. 162(11):6589-95; the contents of each of which are hereinincorporated by reference in their entirety). Furthermore, the polarityof the serine residues improves solubility and prevents aggregationproblems.

In certain embodiments, payload regions of the present disclosure mayencode small and unbranched serine-rich peptide linkers, such as thosedescribed by Huston et al, in U.S. Pat. No. 5,525,491, the contents ofwhich are herein incorporated in their entirety. Polypeptides encoded bythe payload region of the present disclosure, linked by serine-richlinkers, have increased solubility.

In certain embodiments, payload regions of the present disclosure mayencode artificial linkers, such as those described by Whitlow andFilpula in U.S. Pat. No. 5,856,456 and Ladner et al, in U.S. Pat. No.4,946,778, the contents of each of which are herein incorporated bytheir entirety.

In certain embodiments, the payload region encodes at least one G4S3(3×G4S) linker (SEQ ID NO: 70). In certain embodiments, the payloadregion encodes at least one G4S linker (SEQ ID NO: 71). In certainembodiments, the payload region encodes at least one furin site. Incertain embodiments, the payload region encodes at least one T2A linker.In certain embodiments, the payload region encodes at least one F2Alinker. In certain embodiments, the payload region encodes at least oneP2A linker. In certain embodiments, the payload region encodes at leastone IRES sequence. In certain embodiments, the payload region encodes atleast one G4S5 (5×G4S) linker (SEQ ID NO: 69). In certain embodiments,the payload region encodes at least one furin and one 2A linker. Incertain embodiments, the payload region encodes at least one hingeregion. As a non-limiting example, the hinge is a IgG hinge.

In certain embodiments, the linker region may be 1-50, 1-100, 50-100,50-150, 100-150, 100-200, 150-200, 150-250, 200-250, 200-300, 250-300,250-350, 300-350, 300-400, 350-400, 350-450, 400-450, 400-500, 450-500,450-550, 500-550, 500-600, 550-600, 550-650, or 600-650 nucleotides inlength. The linker region may have a length of 1-650 nucleotides (or anyvalue or range therein) or greater than 650. In certain embodiments, thelinker region may be 12 nucleotides in length. In certain embodiments,the linker region may be 18 nucleotides in length. In certainembodiments, the linker region may be 45 nucleotides in length. Incertain embodiments, the linker region may be 54 nucleotides in length.In certain embodiments, the linker region may be 66 nucleotides inlength. In certain embodiments, the linker region may be 75 nucleotidesin length. In certain embodiments, the linker region may be 78nucleotides in length. In certain embodiments, the linker region may be87 nucleotides in length. In certain embodiments, the linker region maybe 108 nucleotides in length. In certain embodiments, the linker regionmay be 153 nucleotides in length. In certain embodiments, the linkerregion may be 198 nucleotides in length. In certain embodiments, thelinker region may be 623 nucleotides in length.

Introns

In certain embodiments, the vector genome includes at least one elementto enhance the transgene target specificity and expression (See e.g.,Powell et al. Viral Expression Cassette Elements to Enhance TransgeneTarget Specificity and Expression in Gene Therapy, 2015; the contents ofwhich are herein incorporated by reference in its entirety) such as anintron. Non-limiting examples of introns include, MVM (67-97 bps), FIXtruncated intron 1 (300 bps), β-globin SD/immunoglobulin heavy chainsplice acceptor (250 bps), adenovirus splice donor/immunoglobin spliceacceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S)(180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230bps).

In certain embodiments, the intron or intron portion may be 100-500nucleotides in length. The intron may have a length of 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490 or 500. The intron may have a length between80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350,80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or400-500.

Stuffer Sequences

In certain embodiments, the viral genome includes at least one elementto improve packaging efficiency and expression, such as a stuffer orfiller sequence. Non-limiting examples of stuffer sequences includealbumin and/or alpha-1 antitrypsin. Any known viral, mammalian, or plantsequence may be manipulated for use as a stuffer sequence.

In certain embodiments, the stuffer or filler sequence may be from about100-3500 nucleotides in length. The stuffer sequence may have a lengthof about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900 or 3000.

miRNA

In certain embodiments, the viral genome includes at least one sequenceencoding a miRNA to reduce the expression of the transgene in a specifictissue, miRNAs and their targeted tissues are well known in the art. Asa non-limiting example, a miR-122 miRNA may be encoded in the viralgenome to reduce the expression of the viral genome in the liver.

Payload

AAV particles of the present disclosure can include, or be producedusing, at least one payload construct which includes at least onepayload region. In certain embodiments, the payload construct includesan expression control sequence operably linked to a payload region. Incertain embodiments, the payload region may be located within a viralgenome, such as the viral genome of a payload construct. At the 5 and/orthe 3′ end of the payload region there may be at least one invertedterminal repeat (ITR). Within the payload region, there may be apromoter region, an intron region and a coding region.

In certain embodiments, a payload construct of the present disclosurecan be a bacmid, also known as a baculovirus plasmid.

In certain embodiments, the payload region of the AAV particle includesone or more nucleic acid sequences encoding a polypeptide or protein ofinterest.

In certain embodiments, the AAV particle includes a viral genome with apayload region comprising nucleic acid sequences encoding more than onepolypeptide of interest. In certain embodiments, a viral genome encodingone or more polypeptides may be replicated and packaged into a viralparticle. A target cell transduced with a viral particle comprising thevector genome may express each of the one or more polypeptides in thesingle target cell.

Where the AAV particle payload region encodes a polypeptide, thepolypeptide may be a peptide, polypeptide or protein. As a non-limitingexample, the payload region may encode at least one therapeutic proteinof interest. The AAV viral genomes encoding polypeptides describedherein may be useful in the fields of human disease, viruses, infectionsveterinary applications and a variety of in vivo and in vitro settings.

In certain embodiments, administration of the formulated AAV particles(which include the viral genome) to a subject will increase theexpression of a protein in a subject. In certain embodiments, theincrease of the expression of the protein will reduce the effects and/orsymptoms of a disease or ailment associated with the polypeptide encodedby the payload.

In certain embodiments, the AAV particle includes a viral genome with apayload region comprising a nucleic acid sequence encoding a protein ofinterest (i.e. a payload protein, therapeutic protein).

In certain embodiments, the payload region comprises a nucleic acidsequence encoding a protein including but not limited to an antibody,Aromatic L-Amino Acid Decarboxylase (AADC), ApoE2, Frataxin, survivalmotor neuron (SMN) protein, glucocermbrosidase, N-sulfoglucosaminesulfohydrolase, N-acetyl-alpha-glucosaminidase, iduronate 2-sulfatase,alpha-L-iduronidase, palmitoyl-protein thioesterase 1, tripeptidylpeptidase 1, battenin, CLN5, CLN6 (linclin), MFSD8. CLN8, aspartoacylase(ASPA), progranulin (GRN), MeCP2, beta-galactosidase (GLB1) and/orgigaxonin (GAN).

In certain embodiments, the AAV particle includes a viral genome with apayload region comprising a nucleic acid sequence encoding any of thedisease-associated proteins (and fragment or variants thereof) describedin any one of the following International Publications: WO2016073693,WO2017023724, WO2018232055, WO2016077687, WO2016077689, WO2018204786,WO2017201258, WO2017201248, WO2018204803, WO2018204797, WO2017189959,WO2017189963, WO2017189964, WO2015191508, WO2016094783, WO20160137949,WO2017075335; the contents of which are each herein incorporated byreference in their entirety.

Amino acid sequences encoded by payload regions of the viral genomes ofthe disclosure may be translated as a whole polypeptide, a plurality ofpolypeptides or fragments of polypeptides, which independently may beencoded by one or more nucleic acids, fragments of nucleic acids orvariants of any of the aforementioned. As used herein, “polypeptide”means a polymer of amino acid residues (natural or unnatural) linkedtogether most often by peptide bonds. The term, as used herein, refersto proteins, polypeptides, and peptides of any size, structure, orfunction. In some instances, the polypeptide encoded is smaller thanabout 50 amino acids and the polypeptide is then termed a peptide. Ifthe polypeptide is a peptide, it will be at least about 2, 3, 4, or atleast 5 amino acid residues long. Thus, polypeptides include geneproducts, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidesand may be associated or linked. The term polypeptide may also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid.

In certain embodiments a “polypeptide variant” is provided. The term“polypeptide variant” refers to molecules which differ in their aminoacid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and in certain embodiments, they will be at least about 80%,or at least about 90% identical (homologous) to a native or referencesequence.

The present disclosure comprises the use of formulated AAV particleswhose vector genomes encode modulatory polynucleotides, e.g., RNA or DNAmolecules as therapeutic agents. Accordingly, the present disclosureprovides vector genomes which encode polynucleotides which are processedinto small double stranded RNA (dsRNA) molecules (small interfering RNA,siRNA, miRNA, pre-miRNA) targeting a gene of interest. The presentdisclosure also provides methods of their use for inhibiting geneexpression and protein production of an allele of the gene of interest,for treating diseases, disorders, and/or conditions.

In certain embodiments, the AAV particle includes a viral genome with apayload region comprising a nucleic acid sequence encoding or includingone or more modulatory polynucleotides. In certain embodiments, the AAVparticle includes a viral genome with a payload region comprising anucleic acid sequence encoding a modulatory polynucleotide of interest.In certain embodiments of the present disclosure, modulatorypolynucleotides, e.g., RNA or DNA molecules, are presented astherapeutic agents. RNA interference mediated gene silencing canspecifically inhibit targeted gene expression.

In certain embodiments, the payload region comprises a nucleic acidsequence encoding a modulatory polynucleotide which interferes with atarget gene expression and/or a target protein production. In certainembodiments, the gene expression or protein production to beinhibited/modified may include but are not limited to superoxidedismutase 1 (SOD1), chromosome 9 open reading frame 72 (C90RF72). TARDNA binding protein (TARDBP), ataxin-3 (ATXN3), huntingtin (HTT),amyloid precursor protein (APP), apolipoprotein E (ApoE),microtubule-associated protein tau (MAPT), alpha-synuclein (SNCA),voltage-gated sodium channel alpha subunit 9 (SCN9A), and/orvoltage-gated sodium channel alpha subunit 10 (SCN10A).

In certain embodiments, the AAV particle includes a viral genome with apayload region comprising a nucleic acid sequence encoding any of themodulatory polynucleotides, RNAi molecules, siRNA molecules, dsRNAmolecules, and/or RNA duplexes described in any one of the followingInternational Publications: WO2016073693, WO2017023724, WO2018232055,WO2016077687, WO2016077689, WO2018204786, WO2017201258, WO2017201248,WO2018204803, WO2018204797, WO2017189959, WO2017189963, WO2017189964,WO2015191508, WO2016094783, WO20160137949, WO2017075335; the contents ofwhich are each herein incorporated by reference in their entirety.

In certain embodiments, a nucleic acid sequence encoding such siRNAmolecules, or a single strand of the siRNA molecules, is inserted intoadeno-associated viral vectors and introduced into cells, specificallycells in the central nervous system.

AAV particles have been investigated for siRNA delivery because ofseveral unique features. Non-limiting examples of the features include(i) the ability to infect both dividing and non-dividing cells; (ii) abroad host range for infectivity, including human cells; (iii) wild-typeAAV has not been associated with any disease and has not been shown toreplicate in infected cells; (iv) the lack of cell-mediated immuneresponse against the vector and (v) the non-integrative nature in a hostchromosome thereby reducing potential for long-term expression.Moreover, infection with AAV particles has minimal influence on changingthe pattern of cellular gene expression (Stilwell and Samulski et al.,Biotechniques, 2003, 34, 148).

In certain embodiments, the encoded siRNA duplex of the presentdisclosure contains an antisense strand and a sense strand hybridizedtogether forming a duplex structure, wherein the antisense strand iscomplementary to the nucleic acid sequence of the targeted gene ofinterest, and wherein the sense strand is homologous to the nucleic acidsequence of the targeted gene of interest. In other aspects, there are0, for 2 nucleotide overhangs at the 3′end of each strand.

The payloads of the formulated AAV particles of the present disclosuremay encode one or more agents which are subject to RNA interference(RNAi) induced inhibition of gene expression. Provided herein areencoded siRNA duplexes or encoded dsRNA that target a gene of interest(referred to herein collectively as “siRNA molecules”). Such siRNAmolecules, e.g., encoded siRNA duplexes, encoded dsRNA or encoded siRNAor dsRNA precursors can reduce or silence gene expression in cells, forexample, astrocytes or microglia, cortical, hippocampal, entorhinal,thalamic, sensory or motor neurons.

RNAi (also known as post-transcriptional gene silencing (PTGS),quelling, or co-suppression) is a post-transcriptional gene silencingprocess in which RNA molecules, in a sequence specific manner, inhibitgene expression, typically by causing the destruction of specific mRNAmolecules. The active components of RNAi are short/small double strandedRNAs (dsRNAs), called small interfering RNAs (siRNAs), that typicallycontain 15-30 nucleotides (e.g., 19 to 25, 19 to 24 or 19-21nucleotides) and 2-nucleotide 3′ overhangs and that match the nucleicacid sequence of the target gene. These short RNA species may benaturally produced in vivo by Dicer-mediated cleavage of larger dsRNAsand they are functional in mammalian cells.

Naturally expressed small RNA molecules, known as microRNAs (miRNAs),elicit gene silencing by regulating the expression of mRNAs. The miRNAscontaining RNA Induced Silencing Complex (RISC) targets mRNAs presentinga perfect sequence complementarity with nucleotides 2-7 in the 5′ regionof the miRNA which is called the seed region, and other base pairs withits 3′ region, miRNA mediated down regulation of gene expression may becaused by cleavage of the target mRNAs, translational inhibition of thetarget mRNAs, or mRNA decay, miRNA targeting sequences are usuallylocated in the 3′ UTR of the target mRNAs. A single miRNA may targetmore than 100 transcripts from various genes, and one mRNA may betargeted by different miRNAs.

siRNA duplexes or dsRNA targeting a specific mRNA may be designed as apayload of an AAV particle and introduced into cells for activating RNAiprocesses. Elbashir et al. demonstrated that 21-nucleotide siRNAduplexes (termed small interfering RNAs) were capable of effectingpotent and specific gene knockdown without inducing immune response inmammalian cells (Elbashir S M et al., Nature, 2001, 411, 494-498). Sincethis initial report, post-transcriptional gene silencing by siRNAsquickly emerged as a powerful tool for genetic analysis in mammaliancells and has the potential to produce novel therapeutics.

The siRNA duplex comprised of a sense strand homologous to the targetmRNA and an antisense strand that is complementary to the target mRNAoffers much more advantage in terms of efficiency for target RNAdestruction compared to the use of the single strand (ss)-siRNAs (e.g.antisense strand RNA or antisense oligonucleotides). In many cases itrequires higher concentration of the ss-siRNA to achieve the effectivegene silencing potency of the corresponding duplex.

In certain embodiments, the siRNA molecules may be encoded in amodulatory polynucleotide which also comprises a molecular scaffold. Asused herein a “molecular scaffold” is a framework or starting moleculethat forms the sequence or structural basis against which to design ormake a subsequent molecule.

In certain embodiments, the modulatory polynucleotide which comprisesthe payload (e.g., siRNA, miRNA or other RNAi agent described herein)includes molecular scaffold which comprises a leading 5′ flankingsequence which may be of any length and may be derived in whole or inpart from wild type microRNA sequence or be completely artificial. A 3′flanking sequence may mirror the 5′ flanking sequence in size andorigin. In certain embodiments, one or both of the 5′ and 3′ flankingsequences are absent.

In certain embodiments, the molecular scaffold may comprise one or morelinkers known in the art. The linkers may separate regions or onemolecular scaffold from another. As a non-limiting example, themolecular scaffold may be polycistronic.

In certain embodiments, the modulatory polynucleotide is designed usingat least one of the following properties: loop variant, seedmismatch/bulge/wobble variant, stem mismatch, loop variant and basalstem mismatch variant, seed mismatch and basal stem mismatch variant,stem mismatch and basal stem mismatch variant, seed wobble and basalstem wobble variant, or a stem sequence variant.

Genome Size

In certain embodiments, the AAV particle which includes a payloaddescribed herein may be single stranded or double stranded vectorgenome. The size of the vector genome may be small, medium, large or themaximum size. Additionally, the vector genome may include a promoter anda polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein may be a small single stranded vector genome. A smallsingle stranded vector genome may be 2.1 to 3.5 kb in size such as about2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,and 3.5 kb in size. As a non-limiting example, the small single strandedvector genome may be 3.2 kb in size. As another non-limiting example,the small single stranded vector genome may be 2.2 kb in size.Additionally, the vector genome may include a promoter and a polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein may be a small double stranded vector genome. A smalldouble stranded vector genome may be 1.3 to 1.7 kb in size such as about1.3, 1.4, 1.5, 1.6, and 1.7 kb in size. As a non-limiting example, thesmall double stranded vector genome may be 1.6 kb in size. Additionally,the vector genome may include a promoter and a polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein e.g., polynucleotide, siRNA or dsRNA, may be a mediumsingle stranded vector genome. A medium single stranded vector genomemay be 3.6 to 4.3 kb in size such as about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2 and 4.3 kb in size. As a non-limiting example, the medium singlestranded vector genome may be 4.0 kb in size. Additionally, the vectorgenome may include a promoter and a polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein may be a medium double stranded vector genome. A mediumdouble stranded vector genome may be 1.8 to 2.1 kb in size such as about1.8, 1.9, 2.0, and 2.1 kb in size. As a non-limiting example, the mediumdouble stranded vector genome may be 2.0 kb in size. Additionally, thevector genome may include a promoter and a polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein may be a large single stranded vector genome. A largesingle stranded vector genome may be 4.4 to 6.0 kb in size such as about4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9 and 6.0 kb in size. As a non-limiting example, the large singlestranded vector genome may be 4.7 kb in size. As another non-limitingexample, the large single stranded vector genome may be 4.8 kb in size.As yet another non-limiting example, the large single stranded vectorgenome may be 6.0 kb in size. Additionally, the vector genome mayinclude a promoter and a polyA tail.

In certain embodiments, the vector genome which includes a payloaddescribed herein may be a large double stranded vector genome. A largedouble stranded vector genome may be 2.2 to 3.0 kb in size such as about2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size. As anon-limiting example, the large double stranded vector genome may be 2.4kb in size. Additionally, the vector genome may include a promoter and apolyA tail.

AAV Serotypes

AAV particles of the present disclosure may include or be derived fromany natural or recombinant AAV serotype. According to the presentdisclosure, the AAV particles may utilize or be based on a serotype orinclude a peptide selected from any of the following: VOY101, VOY201,AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32,AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT,AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T,AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP,AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT,AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST,AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP,AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4),AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3,AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8,AAV9, AAV9.11. AAV9.13, AAV9.16, AAV9.24. AAV9.45, AAV9.47, AAV9.61,AAV9.68. AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1.AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4,AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11. AAV42-12,AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21,AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1,AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48,AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51.AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/rl1.64,AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7,AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2,AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25,AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ.AAV-DJ8, AAVF3, AAVF5. AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70,AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55,AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03,AAVH-I/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5,AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2,AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10. AAVhu,1, AAVhu.13, AAVhu.15, AAVhu.16. AAVhu.17. AAVhu.18. AAVhu.20, AAVhu.21,AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29,AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39,AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48,AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61,AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2,AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R,AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22,AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31. AAVrh.32, AAVrh.33, AAVrh.34,AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49,AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58,AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73,AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVth8R R533A mutant. AAAV,BAAV, caprine AAV, bovine AAV, AAVhE1.1. AAVhEr1.5, AAVhER1.14,AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36,AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36,AAVhER1.23. AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LKO1, AAV-LK02, AAV-LK03,AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10,AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17,AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7,AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b,AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVShuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10,BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48,AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39,AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21,AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true typeAAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAVCBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAVCBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAVCBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAVCBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAVCHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAVCHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3. AAV CKd-4, AAV CKd-6, AAVCKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAVCKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAVCKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAVCKd-N9, AAV CLg-FL, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5. AAVCLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1. AAV CLvI-1, AAV Clv1-10, AAVCLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAVClv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAVCLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAVCLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1. AAV CLv-K1, AAV CLv-K3, AAVCLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAVCLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAVCLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAVCLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAVCSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAVCSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAVCSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355,AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13,AAVFI4/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2,AAVF3/HSC3. AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8,AAVF9/HSC9, AAVrh20, AAVrh32/33, AAVrh39, AAVrh46, AAVrh73, AAVrh74,AAVhu.26, or variants or derivatives thereof.

The AAV-DJ sequence may include two mutations: (1) R587Q where arginine(R: Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2)R590T where arginine (R; Arg) at amino acid 590 is changed to threonine(T; Thr). As another non-limiting example, may include three mutations:(1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine(R: Arg), (2) R587Q where arginine (R: Arg) at amino acid 587 is changedto glutamine (Q; Gln) and (3) R590T where arginine (R; Arg) at aminoacid 590 is changed to threonine (T: Thr).

In certain embodiments, the AAV may be a serotype generated by the AAV9capsid library with mutations in amino acids 390-627 (VP1 numbering) Theserotype and corresponding nucleotide and amino acid substitutions maybe, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A andT1436X: V473D and 1479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C andA1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A,A587V), AAV9.6 (T1231 A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10(A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C. A1769T; T568P, Q590L),AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C,G1713A: L447H), AAV9.16 (A1775T: Q592L), AAV9.24 (T1507C. T1521G;W503R), AAV9.26 (A1337G. A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A),AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T,A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V6061), AAV9.40 (A1694T,E565V), AAV9.41 (A1348T, T1362C: T450S), AAV9.44 (A1684C, A1701T.A1737G; N562H, K567N), AAV9.45 (A1492T. C1804T; N498Y, L602F), AAV9.46(G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A,A1669G, C1745T; S414N, G453D, K557E, T5821), AAV9.48 (C1445T, A1736T:P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53(G1301A, A1405C. C1664T, G181 iT; R134Q, S469R, A555V, G604V), AAV9.54(C1531A, T1609A; L5111, L537M), AAV9.55 (T1605A: F535L), AAV9.58(C1475T. C1579A; T4921, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T:N498I), AAV9.64 (C1531A, A1617T; L511 I), AAV9.65 (CI335T, T1530C,C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A; G481R),AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C. T1468C; S490P),AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R,K5281), AAV9.93 (A1273G, A1421G, A1638C. C1712T, G1732A, A1744T, A1832T;S425G, Q474R. Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T:M559L) and AAV9.95 (T1605A: F535L).

In any of the DNA and RNA sequences referenced and/or described herein,the single letter symbol has the following description: A for adenine; Cfor cytosine; G for guanine; T for thymine; U for Uracil; W for weakbases such as adenine or thymine: S for strong nucleotides such ascytosine and guanine; M for amino nucleotides such as adenine andcytosine: K for keto nucleotides such as guanine and thymine; R forpurines adenine and guanine; Y for pyrimidine cytosine and thymine; Bfor any base that is not A (e.g., cytosine, guanine, and thymine): D forany base that is not C (e.g., adenine, guanine, and thymine): H for anybase that is not G (e.g., adenine, cytosine, and thymine); V for anybase that is not T (e.g., adenine, cytosine, and guanine): N for anynucleotide (which is not a gap); and Z is for zero.

In any of the amino acid sequences referenced and/or described herein,the single letter symbol has the following description: G (Gly) forGlycine; A (Ala) for Alanine: L (Leu) for Leucine; M (Met) forMethionine; F (Phe) for Phenylalanine; W (Trp) for Tryptophan; K (Lys)for Lysine; Q (Gln) for Glutamine; E (Glu) for Glutamic Acid; S (Ser)for Serine: P (Pro) for Proline; V (Val) for Valine; I (Ile) forIsoleucine: C (Cys) for Cysteine; Y (Tyr) for Tyrosine; H (His) forHistidine; R (Arg) for Arginine; N (Asn) for Asparagine; D (Asp) forAspartic Acid; T (Thr) for Threonine; B (Asx) for Aspartic acid orAsparagine; J (Xle) for Leucine or Isoleucine; O (Pyl) for Pyrrolysine;U (Sec) for Selenocysteine; X (Xaa) for any amino acid; and Z (Glx) forGlutamine or Glutamic acid.

In certain embodiments, the AAV serotype may be, or may include asequence, insert, modification or mutation as described in PatentPublications WO2015038958, WO2017100671, WO2016134375, WO2017083722,WO2017015102, WO2017058892, WO2017066764, U.S. Pat. Nos. 9,624,274,9,475,845, US20160369298, US20170145405, the contents of which areherein incorporated by reference in their entirety.

In certain embodiments, the AAV may be a serotype generated byCre-recombination-based AAV targeted evolution (CREATE) as described byDeverman et al., (Nature Biotechnology 34(2):204-209 (2016)), thecontents of which are herein incorporated by reference in theirentirety. In certain embodiments, the AAV serotype may be as describedin Jackson et al (Frontiers in Molecular Neuroscience 9:154 (2016)), thecontents of which are herein incorporated by reference in theirentirety.

In certain embodiments, the AAV serotype is selected for use due to itstropism for cells of the central nervous system. In certain embodiments,the cells of the central nervous system are neurons. In anotherembodiment, the cells of the central nervous system are astrocytes.

In certain embodiments, the AAV serotype is selected for use due to itstropism for cells of the muscle(s).

In certain embodiments, the initiation codon for translation of the AAVVP1 capsid protein may be CTG, TTG, or GTG as described in U.S. Pat. No.8,163,543, the contents of which are herein incorporated by reference inits entirety.

The present disclosure refers to structural capsid proteins (includingVP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsidproteins form an outer protein structural shell (i.e. capsid) of a viralvector such as AAV. VP capsid proteins synthesized from Cappolynucleotides generally include a methionine as the first amino acidin the peptide sequence (Met1), which is associated with the start codon(AUG or ATG) in the corresponding Cap nucleotide sequence. However, itis common for a first-methionine (Met1) residue or generally any firstamino acid (AA1) to be cleaved off after or during polypeptide synthesisby protein processing enzymes such as Met-aminopeptidases. This“Met/AA-clipping” process often correlates with a correspondingacetylation of the second amino acid in the polypeptide sequence (e.g.,alanine, valine, serine, threonine, etc.). Met-clipping commonly occurswith VP1 and VP3 capsid proteins but can also occur with VP2 capsidproteins.

Where the Met/AA-clipping is incomplete, a mixture of one or more (one,two or three) VP capsid proteins including the viral capsid may beproduced, some of which may include a Met1/AA1 amino acid (Met+/AA+) andsome of which may lack a Met1/AA1 amino acid as a result ofMet/AA-clipping (Met−/AA−). For further discussion regardingMet/AA-clipping in capsid proteins, see Jin, et al. Direct LiquidChromatography/Mass Spectrometry Analysis for Complete Characterizationof Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene TherMethods. 2017 Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylationof Cellular Proteins Creates Specific Degradation Signals. Science. 2010Feb. 19, 327(5968): 973-977; the contents of which are each incorporatedherein by reference in their entirety.

According to the present disclosure, references to capsid proteins isnot limited to either clipped (Met−/AA−) or unclipped (Met+/AA+) andmay, in context, refer to independent capsid proteins, viral capsidsincluded of a mixture of capsid proteins, and/or polynucleotidesequences (or fragments thereof) which encode, describe, produce orresult in capsid proteins of the present disclosure. A direct referenceto a “capsid protein” or “capsid polypeptide” (such as VP1, VP2 or VP2)may also include VP capsid proteins which include a Met1/AA1 amino acid(Met+/AA+) as well as corresponding VP capsid proteins which lack theMet1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−).

Further according to the present disclosure, a reference to a specificSEQ ID NO: (whether a protein or nucleic acid) which includes orencodes, respectively, one or more capsid proteins which include aMet1/AA1 amino acid (Met+/AA+) should be understood to teach the VPcapsid proteins which lack the Met1/AA1 amino acid as upon review of thesequence, it is readily apparent any sequence which merely lacks thefirst listed amino acid (whether or not Met1/AA1).

As a non-limiting example, reference to a VP1 polypeptide sequence whichis 736 amino acids in length and which includes a “Met1” amino acid(Met+) encoded by the AUG/ATG start codon may also be understood toteach a VP1 polypeptide sequence which is 735 amino acids in length andwhich does not include the “Met1” amino acid (Met-) of the 736 aminoacid Met+ sequence. As a second non-limiting example, reference to a VP1polypeptide sequence which is 736 amino acids in length and whichincludes an “AA1” amino acid (AA1+) encoded by any NNN initiator codonmay also be understood to teach a VP1 polypeptide sequence which is 735amino acids in length and which does not include the “AA1” amino acid(AA1-) of the 736 amino acid AA1+ sequence.

References to viral capsids formed from VP capsid proteins (such asreference to specific AAV capsid serotypes), can incorporate VP capsidproteins which include a Met1/AA1 amino acid (Met+/AA1+), correspondingVP capsid proteins which lack the Met1/AA1 amino acid as a result ofMet/AA1-clipping (Met−/AA1−), and combinations thereof (Met+/AA1+ andMet−/AA1−).

As a non-limiting example, an AAV capsid serotype can include VP1(Met+/AA1+), VP1 (Met−/AA1−), or a combination of VP1 (Met+/AA1+) andVP1 (Met−/AA1−). An AAV capsid serotype can also include VP3(Met+/AA1+), VP3 (Met−/AA1−), or a combination of VP3 (Met+/AA1+) andVP3 (Met−/AA1−); and can also include similar optional combinations ofVP2 (Met+/AA1) and VP2 (Met−/AA1−).

Introduction into Cells

The encoded siRNA molecules (e.g., siRNA duplexes) of the presentdisclosure may be introduced into cells by being encoded by the vectorgenome of an AAV particle. These AAV particles are engineered tofacilitate the entry into cells that are not readily amendable totransfection/transduction. Also, some synthetic viral vectors possess anability to integrate the shRNA into the cell genome, thereby leading tostable siRNA expression and long-term knockdown of a target gene. Inthis manner, viral vectors are engineered as vehicles for specificdelivery while lacking the deleterious replication and/or integrationfeatures found in wild-type virus.

In certain embodiments, the encoded siRNA molecule is introduced into acell by transfecting, infecting or transducing the cell with an AAVparticle comprising nucleic acid sequences capable of producing thesiRNA molecule when transcribed in the cell. In certain embodiments, thesiRNA molecule is introduced into a cell by injecting into the cell ortissue an AAV particle comprising a nucleic acid sequence capable ofproducing the siRNA molecule when transcribed in the cell.

In certain embodiments, prior to transfection/transduction, an AAVparticle comprising a nucleic acid sequence encoding the siRNA moleculesof the present disclosure may be transfected into cells.

Other methods for introducing AAV particles comprising the nucleic acidsequence for the siRNA molecules described herein may includephotochemical internalization as described in U. S. Patent publicationNo. 20120264807; the content of which is herein incorporated byreference in its entirety.

In certain embodiments, the formulations described herein may contain atleast one AAV particle comprising the nucleic acid sequence encoding thesiRNA molecules described herein. In certain embodiments, the siRNAmolecules may target the gene of interest at one target site. In anotherembodiment, the formulation comprises a plurality of AAV particles, eachAAV particle comprising a nucleic acid sequence encoding a siRNAmolecule targeting the gene of interest at a different target site. Thegene of interest may be targeted at 2, 3, 4, 5 or more than 5 sites.

In certain embodiments, the AAV particles from any relevant species,such as, but not limited to, human, pig, dog, mouse, rat or monkey maybe introduced into cells.

In certain embodiments, the formulated AAV particles may be introducedinto cells or tissues which are relevant to the disease to be treated.

In certain embodiments, the formulated AAV particles may be introducedinto cells which have a high level of endogenous expression of thetarget sequence.

In another embodiment, the formulated AAV particles may be introducedinto cells which have a low level of endogenous expression of the targetsequence.

In certain embodiments, the cells may be those which have a highefficiency of AAV transduction.

In certain embodiments, formulated AAV particles comprising a nucleicacid sequence encoding the siRNA molecules of the present disclosure maybe used to deliver siRNA molecules to the central nervous system (e.g.,U.S. Pat. No. 6,180,613; the contents of which is herein incorporated byreference in its entirety).

In some aspects, the formulated AAV particles comprising a nucleic acidsequence encoding the siRNA molecules of the present disclosure mayfurther comprise a modified capsid including peptides from non-viralorigin. In other aspects, the AAV particle may contain a CNS specificchimeric capsid to facilitate the delivery of encoded siRNA duplexesinto the brain and the spinal cord. For example, an alignment of capnucleotide sequences from AAV variants exhibiting CNS tropism may beconstructed to identify variable region (VR) sequence and structure.

In certain embodiments, the formulated AAV particle comprising a nucleicacid sequence encoding the siRNA molecules of the present disclosure mayencode siRNA molecules which are polycistronic molecules. The siRNAmolecules may additionally comprise one or more linkers between regionsof the siRNA molecules.

In certain embodiments, a formulated AAV particle may comprise at leastone of the modulatory polynucleotides encoding at least one of the siRNAsequences or duplexes described herein.

In certain embodiments, an expression vector may comprise, from ITR toITR recited 5′ to 3′, an ITR, a promoter, an intron, a modulatorypolynucleotide, a polyA sequence and an ITR.

In certain embodiments, the encoded siRNA molecule may be locateddownstream of a promoter in an expression vector such as, but notlimited to, CMV, U6, H1, CBA or a CBA promoter with a SV40 intron.Further, the encoded siRNA molecule may also be located upstream of thepolyadenylation sequence in an expression vector. As a non-limitingexample, the encoded siRNA molecule may be located within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector. As another non-limiting example, the encoded siRNAmolecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10,5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30,20-25, 20-30 or 25-30 nucleotides downstream from the promoter and/orupstream of the polyadenylation sequence in an expression vector. As anon-limiting example, the encoded siRNA molecule may be located withinthe first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or morethan 25% of the nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located with thefirst 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector.

In certain embodiments, the encoded siRNA molecule may be locatedupstream of the polyadenylation sequence in an expression vector.Further, the encoded siRNA molecule may be located downstream of apromoter such as, but not limited to, CMV. U6, CBA or a CBA promoterwith a SV40 intron in an expression vector. As a non-limiting example,the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more than 30 nucleotides downstream from the promoterand/or upstream of the polyadenylation sequence in an expression vector.As another non-limiting example, the encoded siRNA molecule may belocated within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20,5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25,20-30 or 25-30 nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anon-limiting example, the encoded siRNA molecule may be located withinthe first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or morethan 25% of the nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located with thefirst 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector.

In certain embodiments, the encoded siRNA molecule may be located in ascAAV.

In certain embodiments, the encoded siRNA molecule may be located in anssAAV.

In certain embodiments, the encoded siRNA molecule may be located nearthe 5′ end of the flip ITR in an expression vector. In anotherembodiment, the encoded siRNA molecule may be located near the 3′ end ofthe flip ITR in an expression vector. In yet another embodiment, theencoded siRNA molecule may be located near the 5′ end of the flop ITR inan expression vector. In yet another embodiment, the encoded siRNAmolecule may be located near the 3′ end of the flop ITR in an expressionvector. In certain embodiments, the encoded siRNA molecule may belocated between the 5′ end of the flip ITR and the 3′ end of the flopITR in an expression vector. In certain embodiments, the encoded siRNAmolecule may be located between (e.g., half-way between the 5′ end ofthe flip ITR and 3′ end of the flop ITR or the 3′ end of the flop ITRand the 5′ end of the flip ITR), the 3′ end of the flip ITR and the 5′end of the flip ITR in an expression vector. As a non-limiting example,the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more than 30 nucleotides downstream from the 5′ or 3′end of an ITR (e.g., Flip or Flop ITR) in an expression vector. As anon-limiting example, the encoded siRNA molecule may be located within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotidesupstream from the 5′ or 3′ end of an ITR (e.g., Flip or Flop ITR) in anexpression vector. As another non-limiting example, the encoded siRNAmolecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10,5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30,20-25, 20-30 or 25-30 nucleotides downstream from the 5′ or 3′ end of anITR (e.g., Flip or Flop ITR) in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 upstreamfrom the 5′ or 3′ end of an ITR (e.g., Flip or Flop ITR) in anexpression vector. As a non-limiting example, the encoded siRNA moleculemay be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25% or more than 25% of the nucleotides upstream from the 5′or 3′ end of an ITR (e.g., Flip or Flop ITR) in an expression vector. Asanother non-limiting example, the encoded siRNA molecule may be locatedwith the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15, 5-20%,5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream fromthe 5′ or 3′ end of an ITR (e.g., Flip or Flop ITR) in an expressionvector.

In certain embodiments, AAV particle comprising the nucleic acidsequence for the siRNA molecules of the present disclosure may beformulated for CNS delivery. Agents that cross the brain blood barriermay be used. For example, some cell penetrating peptides that can targetsiRNA molecules to the brain blood barrier endothelium may be used toformulate the siRNA duplexes targeting the gene of interest.

In certain embodiments, the formulated AAV particle comprising a nucleicacid sequence encoding the siRNA molecules of the present disclosure maybe administered directly to the CNS. As a non-limiting example, thevector comprises a nucleic acid sequence encoding the siRNA moleculestargeting the gene of interest.

In specific embodiments, compositions of formulated AAV particlescomprising a nucleic acid sequence encoding the siRNA molecules of thepresent disclosure may be administered in a way which facilitates thevectors or siRNA molecule to enter the central nervous system andpenetrate into motor neurons.

In certain embodiments, the formulated AAV particle may be administeredto a subject (e.g., to the CNS of a subject via intrathecaladministration) in a therapeutically effective amount for the siRNAduplexes or dsRNA to target the motor neurons and astrocytes in thespinal cord and/or brain stem. As a non-limiting example, the siRNAduplexes or dsRNA may reduce the expression of a protein or mRNA.

II. AAV Production General Viral Production Process

Viral production cells for the production of rAAV particles generallyinclude mammalian cell types. However, mammalian cells present severalcomplications to the large-scale production of rAAV particles, includinggeneral low yield of viral-particles-per-replication-cell as well ashigh risks for undesirable contamination from other mammalianbiomaterials in the viral production cell. As a result, insect cellshave become an alternative vehicle for large-scale production of rAAVparticles.

AAV production systems using insect cells also present a range ofcomplications. For example, high-yield production of rAAV particlesoften requires a lower expression of Rep78 compared to Rep52.Controlling the relative expression of Rep78 and Rep52 in insect cellsthus requires carefully designed control mechanisms within the Repoperon. These control mechanisms can include individually engineeredinsect cell promoters, such as ΔIE1 promoters for Rep78 and PolHpromoters for Rep52, or the division of the Rep-encoding nucleotidesequences onto independently engineered sequences or constructs.However, implementation of these control mechanisms often leads toreduced rAAV particle yield or to structurally unstable virions.

In another example, production of rAAV particles requires VP1, VP2 andVP3 proteins which assemble to form the AAV capsid. High-yieldproduction of rAAV particles requires adjusted ratios of VP1, VP2 andVP3, which should generally be around 1:1:10, respectively, but can varyfrom 1-2 for VP1 and/or 1-2 for VP2, relative to 10 VP3 copies. Thisratio is important for the quality of the capsid, as too much VP1destabilizes the capsid and too little VP1 will decrease the infectivityof the virus.

Wild type AAV use a deficient splicing method to control VP1 expression;a weak start codon (ACG) with special surrounding (“Kozak” sequence) tocontrol VP2; and a standard start codon (ATG) for VP3 expression.However, in some baculovirus systems, the mammalian splicing sequencesare not always recognized and unable to properly control the productionof VP1. VP2 and VP3. Consequently, neighboring nucleotides and the ACGstart sequence from VP2 can be used to drive capsid protein production.Unfortunately, for most of the AAV serotypes, this method creates acapsid with a lower ratio of VP1 compared to VP2 (<1 relative to 10 VP3copies). To more effectively control the production of VP proteins,non-canonical or start codons have been used, like TTG, GTG or CTG.However, these start codons are considered suboptimal by those in theart relative to the wild type ATG or ACG start codons (See, WO2007046703and WO2007148971, the contents of which are incorporated herein byreference in their entirety).

In another example, production of rAAV particles using a baculovirus/Sf9system generally requires the widely used bacmid-based BaculovirusExpression Vector System (BEVs), which are not optimized for large-scaleAAV production. Aberrant proteolytic degradation of viral proteins inthe bacmid-based BEVs is an unexpected issue, precluding the reliablelarge-scale production of AAV capsid proteins using the baculovirus/Sf9system.

There is continued need for methods and systems which allow foreffective and efficient large scale (commercial) production of rAAVparticles in mammalian and insect cells.

The details of one or more embodiments of the present disclosure are setforth in the accompanying description below. Other features, objects,and advantages of the present disclosure will be apparent from thedescription, drawings, and the claims. In the description, the singularforms also include the plural unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this present disclosure belongs. Inthe case of conflict with disclosures incorporated by reference, thepresent express description will control.

In certain embodiments, the constructs, polynucleotides, polypeptides,vectors, serotypes, capsids formulations, or particles of the presentdisclosure may be, may include, may be modified by, may be used by, maybe used for, may be used with, or may be produced with any sequence,element, construct, system, target or process described in one of thefollowing International Publications: WO2016073693, WO2017023724,WO2018232055, WO2016077687, WO2016077689, WO2018204786. WO2017201258.WO2017201248, WO2018204803, WO2018204797, WO2017189959, WO2017189963,WO2017189964, WO2015191508, WO2016094783, WO20160137949, WO2017075335;the contents of which are each herein incorporated by reference in theirentirety.

AAV production of the present disclosure includes processes and methodsfor producing AAV particles and viral vectors which can contact a targetcell to deliver a payload, e.g. a recombinant viral construct, whichincludes a nucleotide encoding a payload molecule. In certainembodiments, the viral vectors are adeno-associated viral (AAV) vectorssuch as recombinant adeno-associated viral (rAAV) vectors. In certainembodiments, the AAV particles are adeno-associated viral (AAV)particles such as recombinant adeno-associated viral (rAAV) particles.

The present disclosure provides methods of producing AAV particles orviral vectors by (a) contacting a viral production cell with one or moreviral expression constructs encoding at least one chimeric capsidprotein, and one or more payload construct vectors, wherein said payloadconstruct vector includes a payload construct encoding a payloadmolecule selected from the group consisting of a transgene, apolynucleotide encoding protein, and a modulatory nucleic acid; (b)culturing said viral production cell under conditions such that at leastone AAV particle or viral vector is produced, and (c) isolating said atleast one AAV particle or viral vector.

In these methods a viral expression construct may encode at least onestructural protein and/or at least one non-structural protein. Thestructural protein may include any of the native or wild type capsidproteins VP1, VP2 and/or VP3 or a chimeric protein. The non-structuralprotein may include any of the native or wild type Rep78, Rep68, Rep52and/or Rep40 proteins or a chimeric protein.

In certain embodiments, contacting occurs via transient transfection,viral transduction and/or electroporation.

In certain embodiments, the viral production cell is selected from thegroup consisting of a mammalian cell and an insect cell. In certainembodiments, the insect cell includes a Spodoptera frugiperda insectcell. In certain embodiments, the insect cell includes a Sf9 insectcell. In certain embodiments, the insect cell includes a Sf21 insectcell.

The payload construct vector of the present disclosure may include atleast one inverted terminal repeat (ITR) and may include mammalian DNA.

Also provided are AAV particles and viral vectors produced according tothe methods described herein.

The AAV particles of the present disclosure may be formulated as apharmaceutical composition with one or more acceptable excipients.

In certain embodiments, an AAV particle or viral vector may be producedby a method described herein.

In certain embodiments, the AAV particles may be produced by contactinga viral production cell (e.g., an insect cell or a mammalian cell) withat least one viral expression construct encoding at least one capsidprotein and at least one payload construct vector. The viral productioncell may be contacted by transient transfection, viral transductionand/or electroporation. The payload construct vector may include apayload construct encoding a payload molecule such as, but not limitedto, a transgene, a polynucleotide encoding protein, and a modulatorynucleic acid. The viral production cell can be cultured under conditionssuch that at least one AAV particle or viral vector is produced,isolated (e.g., using temperature-induced lysis, mechanical lysis and/orchemical lysis) and/or purified (e.g., using filtration, chromatographyand/or immunoaffinity purification). As a non-limiting example, thepayload construct vector may include mammalian DNA.

In certain embodiments, the AAV particles are produced in an insect cell(e.g., Spodoptera frugiperda (Sf9) cell) using the method describedherein. As a non-limiting example, the insect cell is contacted usingviral transduction which may include baculoviral transduction.

In another embodiment, the AAV particles are produced in a mammaliancell using the method described herein. As a non-limiting example, themammalian cell is contacted using transient transfection.

In certain embodiments, the viral expression construct may encode atleast one structural protein and at least one non-structural protein. Asa non-limiting example, the structural protein includes VP1, VP2 and/orVP3. As another non-limiting example, the non-structural proteinincludes Rep78. Rep68, Rep52 and/or Rep40.

In certain embodiments, the AAV particle production method describedherein produces greater than 10¹, greater than 10², greater than 10³,greater than 10⁴ or greater than 10⁵ AAV particles in a viral productioncell.

In certain embodiments, a process of the present disclosure includesproduction of viral particles in a viral production cell using a viralproduction system which includes at least one viral expression constructand at least one payload construct. The at least one viral expressionconstruct and at least one payload construct can be co-transfected (e.g.dual transfection, triple transfection) into a viral production cell.The transfection is completed using standard molecular biologytechniques known and routinely performed by a person skilled in the art.The viral production cell provides the cellular machinery necessary forexpression of the proteins and other biomaterials necessary forproducing the AAV particles, including Rep proteins which replicate thepayload construct and Cap proteins which assemble to form a capsid thatencloses the replicated payload constructs. The resulting AAV particleis extracted from the viral production cells and processed into apharmaceutical preparation for administration.

Once administered, the AAV particles contacts a target cell and entersthe cell in an endosome. The AAV particle releases from the endosome andsubsequently contacts the nucleus of the target cell to deliver thepayload construct. The payload construct, e.g. recombinant viralconstruct, is delivered to the nucleus of the target cell wherein thepayload molecule encoded by the payload construct may be expressed.

In certain embodiments, the process for production of viral particlesutilizes seed cultures of viral production cells that include one ormore baculoviruses (e.g., a Baculoviral Expression Vector (BEV) or abaculovirus infected insect cell (BIIC) that has been transfected with aviral expression construct and a payload construct vector). In certainembodiments, the seed cultures are harvested, divided into aliquots andfrozen, and may be used at a later time point to initiate an infectionof a naïve population of production cells.

Large scale production of AAV particles may utilize a bioreactor. Theuse of a bioreactor allows for the precise measurement and/or control ofvariables that support the growth and activity of viral production cellssuch as mass, temperature, mixing conditions (impellor RPM or waveoscillation). CO₂ concentration, O₂ concentration, gas sparge rates andvolumes, gas overlay rates and volumes, pH, Viable Cell Density (VCD),cell viability, cell diameter, and/or optical density (OD). In certainembodiments, the bioreactor is used for batch production in which theentire culture is harvested at an experimentally determined time pointand AAV particles are purified. In another embodiment, the bioreactor isused for continuous production in which a portion of the culture isharvested at an experimentally determined time point for purification ofAAV particles, and the remaining culture in the bioreactor is refreshedwith additional growth media components.

AAV viral particles can be extracted from viral production cells in aprocess which includes cell lysis, clarification, sterilization andpurification. Cell lysis includes any process that disrupts thestructure of the viral production cell, thereby releasing AAV particles.In certain embodiments cell lysis may include thermal shock, chemical,or mechanical lysis methods. Clarification can include the grosspurification of the mixture of lysed cells, media components, and AAVparticles. In certain embodiments, clarification includes centrifugationand/or filtration, including but not limited to depth end, tangentialflow, and/or hollow fiber filtration.

The end result of viral production is a purified collection of AAVparticles which include two components: (1) a payload construct (e.g. arecombinant viral genome construct) and (2) a viral capsid.

In certain embodiments, a viral production system or process of thepresent disclosure includes steps for producing baculovirus infectedinsect cells (BIICs) using Viral Production Cells (VPC) and plasmidconstructs. Viral Production Cells (VPCs) from a Cell Bank (CB) arethawed and expanded to provide a target working volume and VPCconcentration. The resulting pool of VPCs is split into a Rep/Cap VPCpool and a Payload VPC pool. One or more Rep/Cap plasmid constructs(viral expression constructs) are processed into Rep/Cap Bacmidpolynucleotides and transfected into the Rep/Cap VPC pool. One or morePayload plasmid constructs (payload constructs) are processed intoPayload Bacmid polynucleotides and transfected into the Payload VPCpool. The two VPC pools are incubated to produce P1 Rep/Cap BaculoviralExpression Vectors (BEVs) and P1 Payload BEVs. The two BEV pools areexpanded into a collection of Plaques, with a single Plaque beingselected for Clonal Plaque (CP) Purification (also referred to as SinglePlaque Expansion). The process can include a single CP Purification stepor can include multiple CP Purification steps either in series orseparated by other processing steps. The one-or-more CP Purificationsteps provide a CP Rep/Cap BEV pool and a CP Payload BEV pool. These twoBEV pools can then be stored and used for future production steps, orthey can be then transfected into VPCs to produce a Rep/Cap BIIC pooland a Payload BIIC pool.

In certain embodiments, a viral production system or process of thepresent disclosure includes steps for producing AAV particles usingViral Production Cells (VPC) and baculovirus infected insect cells(BIICs). Viral Production Cells (VPCs) from a Cell Bank (CB) are thawedand expanded to provide a target working volume and VPC concentration.The working volume of Viral Production Cells is seeded into a ProductionBioreactor and can be further expanded to a working volume of 200-2000 Lwith a target VPC concentration for B11C infection. The working volumeof VPCs in the Production Bioreactor is then co-infected with Rep/CapBIICs and Payload BIICs, with a target VPC:BIIC ratio and a targetBIIC:BIIC ratio. VCD infection can also utilize BEVs. The co-infectedVPCs are incubated and expanded in the Production Bioreactor to producea bulk harvest of AAV particles and VPCs.

Viral Expression Construct

The viral production system of the present disclosure includes one ormore viral expression constructs which can be transfected/transducedinto a viral production cell. In certain embodiments, the viralexpression includes a protein-coding nucleotide sequence and at leastone expression control sequence for expression in a viral productioncell. In certain embodiments, the viral expression includes aprotein-coding nucleotide sequence operably linked to least oneexpression control sequence for expression in a viral production cell.In certain embodiments, the viral expression construct containsparvoviral genes under control of one or more promoters. Parvoviralgenes can include nucleotide sequences encoding non-structural AAVreplication proteins, such as Rep genes which encode Rep52, Rep40, Rep68or Rep78 proteins. Parvoviral genes can include nucleotide sequencesencoding structural AAV proteins, such as Cap genes which encode VP1,VP2 and VP3 proteins.

In certain embodiments, a viral expression construct can include aRep52-coding region; a Rep52-coding region is a nucleotide sequencewhich includes a Rep52 nucleotide sequence encoding a Rep52 protein. Incertain embodiments, a viral expression construct can include aRep78-coding region; a Rep78-coding region is a nucleotide sequencewhich includes a Rep78 nucleotide sequence encoding a Rep78 protein. Incertain embodiments, a viral expression construct can include aRep40-coding region; a Rep40-coding region is a nucleotide sequencewhich includes a Rep40 nucleotide sequence encoding a Rep40 protein. Incertain embodiments, a viral expression construct can include aRep68-coding region; a Rep68-coding region is a nucleotide sequencewhich includes a Rep68 nucleotide sequence encoding a Rep68 protein.

In certain embodiments, a viral expression construct can include aVP-coding region; a VP-coding region is a nucleotide sequence whichincludes a VP nucleotide sequence encoding VP1, VP2, VP3, or acombination thereof. In certain embodiments, a viral expressionconstruct can include a VP1-coding region; a VP1-coding region is anucleotide sequence which includes a VP1 nucleotide sequence encoding aVP1 protein. In certain embodiments, a viral expression construct caninclude a VP2-coding region; a VP2-coding region is a nucleotidesequence which includes a VP2 nucleotide sequence encoding a VP2protein. In certain embodiments, a viral expression construct caninclude a VP3-coding region; a VP3-coding region is a nucleotidesequence which includes a VP3 nucleotide sequence encoding a VP3protein.

Structural VP proteins, VP1, VP2, and VP3, and non-structural proteins,Rep52 and Rep78, of the viral expression construct can be encoded in asingle open reading frame regulated by utilization of both alternativesplice acceptor and non-canonical translational initiation codons. BothRep78 and Rep52 can be translated from a single transcript: Rep78translation initiates at a first start codon (AUG or non-AUG) and Rep52translation initiates from a Rep52 start codon (e.g. AUG) within theRep78 sequence. Rep78 and Rep52 can also be translated from separatetranscripts with independent start codons. The Rep52 initiation codonswithin the Rep78 sequence can be mutated, modified or removed, such thatprocessing of the modified Rep78 sequence will not produce Rep52proteins.

VP1, VP2 and VP3 can be transcribed and translated from a singletranscript wherein both in-frame and out-of-frame ATG tripletspreventing translation initiation at a position between the VP1 and VP2start codons are eliminated. In certain embodiments, VP1 can betranscribed and translated independently from VP2 and VP3 from atranscript which encodes only for VP1, and not for VP2 or VP3. As useherein, the terms “encodes only for VP1” or “encodes for VP1, but notVP2 or VP3” refer to a nucleotide sequence or transcript which encodesfor a VP1 capsid protein and which lacks the necessary codons within theVP1 sequence (deleted or mutated) for transcription or translation ofVP2 and VP3 from the same sequence, or which includes additional codonswithin the VP1 sequence which prevent transcription or translation ofVP2 and VP3 from the same sequence.

The viral production system of the present disclosure is not limited bythe viral expression vector used to introduce the parvoviral functionsinto the virus replication cell. The presence of the viral expressionconstruct in the virus replication cell need not be permanent. The viralexpression constructs can be introduced by any means known, for exampleby chemical treatment of the cells, electroporation, or infection.

Viral expression constructs of the present disclosure may include anycompound or formulation, biological or chemical, which facilitatestransformation, transfection, or transduction of a cell with a nucleicacid. Exemplary biological viral expression constructs include plasmids,linear nucleic acid molecules, and recombinant viruses includingbaculovirus. Exemplary chemical vectors include lipid complexes. Viralexpression constructs are used to incorporate nucleic acid sequencesinto virus replication cells in accordance with the present disclosure.(O'Reilly, David R, Lois K. Miller, and Verne A. Luckow. Baculovirusexpression vectors: a laboratory manual. Oxford University Press,1994.); Maniatis et al., eds. Molecular Cloning. CSH Laboratory, NY,N.Y. (1982); and Philiport and Scluber, eds. Liposomes as tools in BasicResearch and Industry. CRC Press, Ann Arbor, Mich. (1995), the contentsof each of which are herein incorporated by reference in its entirety.

In certain embodiments, the viral expression construct is an AAVexpression construct which includes one or more nucleotide sequencesencoding non-structural AAV replication proteins, structural AAV capsidproteins, or a combination thereof.

In certain embodiments, the viral expression construct of the presentdisclosure may be a plasmid vector. In certain embodiments, the viralexpression construct of the present disclosure may be a baculoviralconstruct.

The present disclosure is not limited by the number of viral expressionconstructs employed to produce AAV particles or viral vectors. Incertain embodiments, one, two, three, four, five, six, or more viralexpression constructs can be employed to produce AAV particles in viralproduction cells in accordance with the present disclosure. In onenon-limiting example, five expression constructs may individually encodeAAV VP1, AAV VP2, AAV VP3, Rep52, Rep78, and with an accompanyingpayload construct comprising a payload polynucleotide and at least oneAAV ITR. In another embodiment, expression constructs may be employed toexpress, for example, Rep52 and Rep40, or Rep78 and Rep 68. Expressionconstructs may include any combination of VP1, VP2, VP3, Rep52/Rep40,and Rcp78/Rep68 coding sequences.

In certain embodiments of the present disclosure, a viral expressionconstruct may be used for the production of an AAV particles in insectcells. In certain embodiments, modifications may be made to the wildtype AAV sequences of the capsid and/or rep genes, for example toimprove attributes of the viral particle, such as increased infectivityor specificity, or to enhance production yields.

In certain embodiments, the viral expression construct can include oneor more expression control sequence between protein-coding nucleotidesequences. In certain embodiments, an expression control region caninclude an IRES sequence region which includes an IRES nucleotidesequence encoding an internal ribosome entry sight (IRES). The internalribosome entry sight (IRES) can be selected from the group consistingor: FMDV-IRES from Foot-and-Mouth-Disease virus, EMCV-IRES fromEncephalomyocarditis virus, and combinations thereof.

In certain embodiments, an expression control region can include a 2Asequence region which comprises a 2A nucleotide sequence encoding aviral 2A peptide. A viral 2A sequence is a relatively short(approximately 20 amino acids) sequence which contains a consensussequence of: Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO: 72). Thesequence allows for co-translation of multiple polypeptides within asingle open reading frame (ORF). As the ORF is translated, glycine andproline residues with the 2A sequence prevent the formation of a normalpeptide bond, which results in ribosomal “skipping” and “self-cleavage”within the polypeptide chain. The viral 2A peptide can be selected fromthe group consisting of: F2A from Foot-and-Mouth-Disease virus, T2A fromThosea asigna virus, E2A from Equine rhinitis A virus, P2A from porcineteschovirus-1, BmCPV2A from cytoplasmic polyhedrosis virus, BmIFV 2Afrom B. mori flacherie virus, and combinations thereof.

In certain embodiments, the viral expression construct may contain anucleotide sequence which includes start codon region, such as asequence encoding AAV capsid proteins which include one or more startcodon regions. In certain embodiments, the start codon region can bewithin an expression control sequence. The start codon can be ATG or anon-ATG codon (i.e., a suboptimal start codon where the start codon ofthe AAV VP1 capsid protein is a non-ATG).

In certain embodiments, the viral expression construct used for AAVproduction may contain a nucleotide sequence encoding the AAV capsidproteins where the initiation codon of the AAV VP1 capsid protein is anon-ATG, i.e., a suboptimal initiation codon, allowing the expression ofa modified ratio of the viral capsid proteins in the production system,to provide improved infectivity of the host cell. In a non-limitingexample, a viral construct vector may contain a nucleic acid constructcomprising a nucleotide sequence encoding AAV VP1, VP2, and VP3 capsidproteins, wherein the initiation codon for translation of the AAV VP1capsid protein is CTG, TTG, or GTG, as described in U.S. Pat. No.8,163,543, the contents of which are herein incorporated by reference inits entirety.

In certain embodiments, the viral expression construct of the presentdisclosure may be a plasmid vector or a baculoviral construct thatencodes the parvoviral rep proteins for expression in insect cells. Incertain embodiments, a single coding sequence is used for the Rep78 andRep52 proteins, wherein start codon for translation of the Rep78 proteinis a suboptimal start codon, selected from the group consisting of ACG,TTG, CTG and GTG, that effects partial exon skipping upon expression ininsect cells, as described in U.S. Pat. No. 8,512,981, the contents ofwhich are herein incorporated by reference in their entirety, forexample to promote less abundant expression of Rep78 as compared toRep52, which may in that it promotes high vector yields.

In certain embodiments, the viral expression construct may be a plasmidvector or a baculoviral construct for the expression in insect cellsthat contains repeating codons with differential codon biases, forexample to achieve improved ratios of Rep proteins, e.g. Rep78 and Rep52thereby improving large scale (commercial) production of viralexpression construct and/or payload construct vectors in insect cells,as taught in U.S. Pat. No. 8,697,417, the contents of which are hereinincorporated by reference in their entirety.

In another embodiment, improved ratios of rep proteins may be achievedusing the method and constructs described in U.S. Pat. No. 8,642,314,the contents of which are herein incorporated by reference in theirentirety.

In certain embodiments, the viral expression construct may encode mutantparvoviral Rep polypeptides which have one or more improved propertiesas compared with their corresponding wild type Rep polypeptide, such asthe preparation of higher virus titers for large scale production.Alternatively, they may be able to allow the production ofbetter-quality viral particles or sustain more stable production ofvirus. In a non-limiting example, the viral expression construct mayencode mutant Rep polypeptides with a mutated nuclear localizationsequence or zinc finger domain, as described in Patent Application US20130023034, the contents of which are herein incorporated by referencein their entirety.

In certain embodiments, the viral expression construct may encode thecomponents of a Parvoviral capsid with incorporated Gly-Ala repeatregion, which may function as an immune invasion sequence, as describedin US Patent Application 20110171262, the contents of which are hereinincorporated by reference in its entirety.

In certain embodiments of the present disclosure, a viral expressionconstruct may be used for the production of AAV particles in insectcells. In certain embodiments, modifications may be made to the wildtype AAV sequences of the capsid and/or rep genes, for example toimprove attributes of the viral particle, such as increased infectivityor specificity, or to enhance production yields.

In certain embodiments, a VP-coding region encodes one or more AAVcapsid proteins of a specific AAV serotype. The AAV serotypes forVP-coding regions can be the same or different. In certain embodiments,a VP-coding region can be codon optimized. In certain embodiments, aVP-coding region or nucleotide sequence can be codon optimized for amammal cell. In certain embodiments, a VP-coding region or nucleotidesequence can be codon optimized for an insect cell. In certainembodiments, a VP-coding region or nucleotide sequence can be codonoptimized for a Spodoptera frugiperda cell. In certain embodiments, aVP-coding region or nucleotide sequence can be codon optimized for Sf9or Sf21 cell lines.

In certain embodiments, a nucleotide sequence encoding one or more VPcapsid proteins can be codon optimized to have a nucleotide homologywith the reference nucleotide sequence of less than 100%. In certainembodiments, the nucleotide homology between the codon-optimized VPnucleotide sequence and the reference VP nucleotide sequence is lessthan 100%, less than 99%, less than 98%, less than 97%, less than 96%,less than 95%, less than 94%, less than 93%, less than 92%, less than91%, less than 90%, less than 89%, less than 88%, less than 87%, lessthan 86%, less than 85%, less than 84%, less than 83%, less than 82%,o,less than 81%, less than 80%, less than 78%, less than 76%, less than74%, less than 72%, less than 70%, less than 68%, less than 66%, lessthan 64%, less than 62%, less than 60%, less than 55%, less than 50%,and less than 40%.

In certain embodiments, viral expression constructs may be used that aretaught in U.S. Pat. Nos. 8,512,981, 8,163,543, 8,697,417, 8,642,314. USPatent Publication Nos. US20130296532, US20110119777, US20110136227.US20110171262, US20130023034, International Patent Application Nos.PCT/NL2008/050613, PCT/NL2009/050076, PCT/NL2009/050352,PCT/NL2011/050170, PCT/NL2012/050619 and U.S. patent application Ser.No. 14/149,953, the contents of each of which are herein incorporated byreference in their entirety.

In certain embodiments, the viral expression construct of the presentdisclosure may be derived from viral expression constructs taught inU.S. Pat. Nos. 6,468,524, 6,984,517, 7,479,554, 6,855,314, 7,271,002,6,723,551, US Patent Publication No. 20140107186, U.S. Ser. No.09/717,789, U.S. Ser. No. 11/936,394, U.S. Ser. No. 14/004,379, EuropeanPatent Application EP1082413, EP2500434, EP 2683829, EP1572893 andInternational Patent Application PCT/US99/11958, PCT/US01/09123,PCT/EP2012/054303, and PCT/US2002/035829 the contents of each of whichare herein incorporated by reference in its entirety.

In certain embodiments, the viral expression construct may includesequences from Simian species. In certain embodiments, the viralexpression construct may contain sequences, including but not limited tocapsid and rep sequences from International Patent ApplicationsPCT/US1997/015694, PCT/US2000/033256, PCT/US2002/019735,PCT/US2002/033645, PCT/US2008/013067, PCT/US2008/013066,PCT/US2008/013065, PCT/US2009/062548, PCT/US2009/001344,PCT/US2010/036332, PCT/US2011/061632, PCT/US2013/041565. U.S. Ser. Nos.13/475,535, 13/896,722, 10/739,096, 14/073,979, US Patent PublicationNos. US20010049144, US20120093853, US20090215871, US20040136963,US20080219954, US20040171807, US20120093778, US20080090281,US20050069866, US20100260799. US20100247490,US20140044680,US20100254947, US20110223135, US20130309205, US20120189582,US20130004461, US20130315871, U.S. Pat. Nos. 6,083,716, 7,838,277,7,344,872, 8,603,459, 8,105,574, 7,247,472, 8,231,880, 8,524,219,8,470,310, European Patent Application Nos. EP2301582, EP2286841,EP1944043, EP1453543, EP1409748, EP2463362, EP2220217, EP2220241,EP2220242, EP2350269, EP2250255, EP2435559, EP2643465, EP1409748,EP2325298, EP1240345, the contents of each of which is hereinincorporated by reference in its entirety.

In certain embodiments, viral expression constructs of the presentdisclosure may include one or more nucleotide sequence from one or moreviral construct described in in International Application No.PCT/US2002/025096, PCT/US2002/033629, PCT/US2003/012405. U.S. Ser. Nos.10/291,583, 10/420,284, U.S. Pat. No. 7,319,002, Patent Publication No.US20040191762, US20130045186, US20110263027, US20110151434,US20030138772, US20030207259, European Application No. EP2338900,EP1456419, EP1310571, EP1359217, EP1427835, EP2338900, EP1456419,EP1310571. EP1359217 and U.S. Pat. Nos. 7,235,393 and 8,524,446.

In certain embodiments, the viral expression constructs of the presentdisclosure may include sequences or compositions described inInternational Patent Application No. PCT/US1999/025694.PCT/US1999/010096, PCT/US2001/013000, PCT/US2002/25976,PCT/US2002/033631, PCT/US2002/033630, PCT/US2009/041606,PCT/US2012/025550, U.S. Pat. Nos. 8,637,255, 8,637,255, 7,186,552,7,105,345, 6,759,237, 7,056,502, 7,198,951, 8,318,480, 7,790,449,7,282,199, US Patent Publication No. US20130059289, US20040057933,US20040057932, US20100278791, US20080050345, US20080050343,US20080008684, US20060204479, US20040057931, US20040052764,US20030013189, US20090227030, US20080075740, US20080075737,US20030228282, US20130323226, US20050014262, U.S. Ser. No. 14/136,331,U.S. Ser. No. 09/076,369, U.S. Ser. No. 10/738,609, European ApplicationNo. EP2573170, EP1127150, EP2341068, EP1845163, EPI 127150, EP1078096,EP1285078, EP1463805, EP2010178940, US20140004143, EP2359869, EP1453547,EP2341068, and EP2675902, the contents of each of which are hereinincorporated by reference in their entirety.

In certain embodiments, viral expression construct of the presentdisclosure may include one or more nucleotide sequence from one or moreof those described in U.S. Pat. Nos. 7,186,552, 7,105,345, 6,759,237,7,056,502, 7,198,951, 8,318,480, 7,790,449, 7,282,199, US PatentPublication No. US20130059289, US20040057933, US20040057932,US20100278791, US20080050345, US20080050343, US20080008684,US20060204479, US20040057931. US20140004143, US20090227030,US20080075740, US20080075737, US20030228282, US20040052764,US20030013189, US20050014262, US20130323226, U.S. Ser. No. 14/136,331,U.S. Ser. No. 10/738,609, European Patent Application Nos. EP1127150,EP2341068, EP1845163, EP1127150, EP1078096, EP1285078, EP2573170,EP1463805, EP2675902, EP2359869, EP1453547, EP2341068, the contents ofeach of which are incorporated herein by reference in their entirety.

In certain embodiments, the viral expression constructs of the presentdisclosure may include constructs of modified AAVs, as described inInternational Patent Application No. PCT/US1995/014018,PCT/US2000/026449, PCT/US2004/028817, PCT/US2006/013375,PCT/US2007/010056, PCT/US2010/032158, PCT/US2010/050135,PCT/US2011/033596, U.S. patent application Ser. No. 12/473,917, U.S.Ser. No. 08/331,384, US09/670277, U.S. Pat. Nos. 5,871,982, 5,856,152,6,251,677. U.S. Pat. Nos. 6,387,368, 6,399,385, 7,906,111, EuropeanPatent Application No. EP2000103600, European Patent Publication No.EP797678, EP1046711. EP1668143, EP2359866, EP2359865, EP2357010,EP1046711, EP1218035, EP2345731. EP2298926, EP2292780, EP2292779,EP1668143, US20090197338, EP2383346, EP2359867, EP2359866, EP2359865,EP2357010, EP1866422, US20090317417, EP2016174, US Patent PublicationNos. US20110236353, US20070036760, US20100186103, US20120137379, andUS20130281516, the contents of each of which are herein incorporated byreference in their entirety.

In certain embodiments, the viral expression constructs of the presentdisclosure may include one or more constructs described in InternationalApplication Nos. PCT/US1999/004367, PCT/US2004/010965,PCT/US2005/014556, PCT/US2006/009699, PCT/US2010/032943.PCT/US2011/033628, PCT/US2011/033616, PCT/US2012/034355, U.S. Pat. No.8,394,386, EP1742668, US Patent Publication Nos. US20080241189,US20120046349, US20130195801, US20140031418, EP2425000, US20130101558,EP1742668, EP2561075, EP2561073, EP2699688, the contents of each ofwhich is herein incorporated by reference in its entirety.

Expression Control Expression Control Regions

The viral expression constructs of the present disclosure can includeone or more expression control regions encoded by expression controlsequences. In certain embodiments, the expression control sequences arefor expression in a viral production cell, such as an insect cell. Incertain embodiments, the expression control sequences are operablylinked to a protein-coding nucleotide sequence. In certain embodiments,the expression control sequences are operably linked to a VP codingnucleotide sequence or a Rep coding nucleotide sequence.

Herein, the terms “coding nucleotide sequence”, “protein-encoding gene”or “protein-coding nucleotide sequence” refer to a nucleotide sequencethat encodes or is translated into a protein product, such as structuralAAV capsid proteins (VP proteins) or non-structural AAV replicationproteins (Rep proteins). “Operably linked” means that the expressioncontrol sequence is positioned relative to the coding sequence such thatit can promote the expression of the encoded gene product.

“Expression control sequence” refers to a nucleic acid sequence thatregulates the expression of a nucleotide sequence to which it isoperably linked. An expression control sequence is “operably linked” toa nucleotide sequence when the expression control sequence controls andregulates the transcription and/or the translation of the nucleotidesequence. Thus, an expression control sequence can include promoters,enhancers, untranslated regions (UTRs), internal ribosome entry sites(IRES), transcription terminators, a start codon in front of aprotein-encoding gene, splicing signal for introns, and stop codons. Theterm “expression control sequence” is intended to include, at a minimum,a sequence whose presence are engineered to influence expression of anucleotide sequence, and can also include additional advantageouscomponents. For example, leader sequences and fusion partner sequencescan be expression control sequences. The term can also include thedesign of the nucleic acid sequence such that undesirable, potentialinitiation codons in and out of frame, are removed from the sequence. Itcan also include the design of the nucleic acid sequence such thatundesirable potential splice sites are removed. It includes sequences orpolyadenylation sequences (pA) which direct the addition of a polyAtail, i.e., a string of adenine residues at the 3′-end of an mRNA,sequences referred to as polyA sequences. It also can be designed toenhance mRNA stability. Expression control sequences which affect thetranscription and translation stability, e.g., promoters, as well assequences which effect the translation, e.g., Kozak sequences, are knownin insect cells. Expression control sequences can be of such nature asto modulate the nucleotide sequence to which it is operably linked suchthat lower expression levels or higher expression levels are achieved.

In certain embodiments, the expression control sequence can include oneor more promoters. Promoters can include, but are not limited to,baculovirus major late promoters, insect virus promoters, non-insectvirus promoters, vertebrate virus promoters, nuclear gene promoters,chimeric promoters from one or more species including virus andnon-virus elements, and/or synthetic promoters. In certain embodiments,a promoter can be selected from: Op-EI, EI, ΔEI, EI-1, pH, PIO, polH(polyhedron), ΔpolH, Dmhsp70, Hr1, Hsp70, 4×Hsp27 EcRE+minimal Hsp70,IE, IE-1, ΔIE-1. ΔIE, p10, Δp10 (modified variations or derivatives ofp10), p5, p19, p35, p40, p6.9, and variations or derivatives thereof. Incertain embodiments, a promoter can be selected from tissue-specificpromoters, cell-type-specific promoters, cell-cycle-specific promoters,and variations or derivatives thereof. In certain embodiments, apromoter can be selected from: CMV promoter, an alpha 1-antitrypsin(al-AT) promoter, a thyroid hormone-binding globulin promoter, athyroxine-binding globlin (LPS) promoter, an HCR-ApoCII hybrid promoter,an HCR-hAAT hybrid promoter, an albumin promoter, an apolipoprotein Epromoter, an α1-AT+EaIb promoter, a tumor-selective E2F promoter, amononuclear blood IL-2 promoter, and variations or derivatives thereof.In certain embodiments, the promoter is a low-expression promotersequence. In certain embodiments, the promoter is an enhanced-expressionpromoter sequence. In certain embodiments, the promoter can include Repor Cap promoters as described in US Patent Application 20110136227, thecontents of which are herein incorporated by reference in its entirety.

In certain embodiments, a viral expression construct can include thesame promoter in all nucleotide sequences. In certain embodiments, aviral expression construct can include the same promoter in two or morenucleotide sequences. In certain embodiments, a viral expressionconstruct can include a different promoter in two or more nucleotidesequences. In certain embodiments, a viral expression construct caninclude a different promoter in all nucleotide sequences.

In certain embodiments the viral expression construct encodes elementsto improve expression in certain cell types. In a further embodiment,the expression construct may include polh and/or ΔIE-1 insecttranscriptional promoters, CMV mammalian transcriptional promoter,and/or p10 insect specific promoters for expression of a desired gene ina mammalian or insect cell.

More than one expression control sequence can be operably linked to agiven nucleotide sequence. For example, a promoter sequence, atranslation initiation sequence, and a stop codon can be operably linkedto a nucleotide sequence.

In certain embodiments, the viral expression construct may contain anucleotide sequence which includes start codon region, such as asequence encoding AAV capsid proteins which include one or more startcodon regions. In certain embodiments, the start codon region can bewithin an expression control sequence.

The translational start site of eukaryotic mRNA is controlled in part bya nucleotide sequence referred to as a Kozak sequence as described inKozak, M Cell. 1986 Jan. 31:44(2):283-92 and Kozak, M. J Cell Biol. 1989February; 108(2):229-41 the contents of each of which are hereinincorporated by reference in their entirety. Both naturally occurringand synthetic translational start sites of the Kozak form can be used inthe production of polypeptides by molecular genetic techniques. Kozak,M. Mamm Genome. 1996 August:7(8):563-74 the contents of which are hereinincorporated by reference in their entirety. Splice sites are sequenceson an mRNA which facilitate the removal of parts of the mRNA sequencesafter the transcription (formation) of the mRNA. Typically, the splicingoccurs in the nucleus, prior to mRNA transport into a cell's cytoplasm.

The method of the present disclosure is not limited by the use ofspecific expression control sequences. However, when a certainstoichiometry of VP products are achieved (close to 1:1:10 for VP1, VP2,and VP3, respectively) and also when the levels of Rep52 or Rep40 (alsoreferred to as the p19 Reps) are significantly higher than Rep78 orRep68 (also referred to as the p5 Reps), improved yields of AAV inproduction cells (such as insect cells) may be obtained. In certainembodiments, the p5/p19 ratio is below 0.6 more, below 0.4, or below0.3, but always at least 0.03. These ratios can be measured at the levelof the protein or can be implicated from the relative levels of specificmRNAs.

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is 1:1:10(VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is 2:2:10(VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is 2:0:10(VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is1-2:0-2:10 (VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is1-2:1-2:10 (VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is2-3:0-3:10 (VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is2-3:2-3:10 (VP1:VP2:VP3).

In certain embodiments. AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is 3:3:10(VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is3-5:0-5:10 (VP1:VP2:VP3).

In certain embodiments, AAV particles are produced in viral productioncells (such as mammalian or insect cells) wherein all three VP proteinsare expressed at a stoichiometry approaching, about or which is3-5:3-5:10 (VP1:VP2:VP3).

In certain embodiments, the expression control regions are engineered toproduce a VP1:VP2:VP3 ratio selected from the group consisting of: aboutor exactly 1:0:10; about or exactly 1:1:10; about or exactly 2:1:10;about or exactly 2:1:10; about or exactly 2:2:10; about or exactly3:0:10; about or exactly 3:1:10; about or exactly 3:2:10; about orexactly 3:3:10; about or exactly 4:0:10; about or exactly 4:1:10; aboutor exactly 4:2:10; about or exactly 4:3:10, about or exactly 4:4:10;about or exactly 5:5:10; about or exactly 1-2:0-2:10: about or exactly1-2:1-2:10: about or exactly 1-3:0-3:10; about or exactly 1-3:1-3:10:about or exactly 1-4:0-4:10: about or exactly 1-4:1-4:10; about orexactly 1-5:1-5:10; about or exactly 2-3:0-3:10; about or exactly2-3:2-3:10; about or exactly 2-4:2-4:10: about or exactly 2-5:2-5:10;about or exactly 3-4:34:10; about or exactly 3-5:3-5:10; and about orexactly 4-5:4-5:10.

In certain embodiments of the present disclosure, Rep52 or Rep78 istranscribed from the baculoviral derived polyhedron promoter, (polh).Rep52 or Rep78 can also be transcribed from a weaker promoter, forexample a deletion mutant of the IE-1 promoter, the ΔIE-1 promoter, hasabout 20% of the transcriptional activity of that IE-1 promoter. Apromoter substantially homologous to the ΔIE-1 promoter may be used. Inrespect to promoters, a homology of at least 50%, 60%, 70%, 80%, 90% ormore, is considered to be a substantially homologous promoter.

Engineered Untranslated Regions (UIRs)

The present disclosure presents engineered untranslated regions (UTRs),including engineered UTR polynucleotides that function as a 5′ UTR.Engineering the features in untranslated regions (UTRs) can improve thestability and protein production capability of the viral productionconstructs of the present disclosure.

The present disclosure presents viral expression constructs whichinclude an engineered untranslated region (UTR) of the presentdisclosure. In some embodiments, the viral expression construct includesan engineered untranslated region (UTR) of the present disclosure. Insome embodiments, the viral expression construct includes an engineered5′ UTR of the present disclosure.

Natural 5′ UTRs include features which play important roles intranslation initiation. They harbor signatures such as a Kozak sequenceswhich are known to be involved in the process by which the ribosomeinitiates translation of many genes. The present disclosure providesengineered polynucleotide sequences which include at least one 5′ UTRfunction. Such “engineered 5′ UTR polynucleotides” or “engineered 5′UTRs” may also include the start codon of the protein whose expressionis being driven, e.g., a structural AAV capsid protein (VP1, VP2 or VP3)or a non-structural AAV replication protein (Rep78 or Rep52).

According to the present disclosure, the engineered 5′ UTRpolynucleotides may range independently from 15-1,000 nucleotides inlength (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides). Non-UTR sequences may be incorporated into theengineered 5′ UTRs. For example, introns or portions of intronssequences may be incorporated into the polynucleotides of thedisclosure. Incorporation of intronic sequences may also increase AAVserotype protein (e.g., capsid) production.

Leader sequences may be included in the engineered polynucleotides. Suchleader sequences may derive from or be identical to all or a portion ofany AAV serotype selected from those taught herein.

“Features”, when referring to polynucleotides, are defined as distinctnucleic acid sequence-based components of a molecule. Features of thepolynucleotides of the present disclosure include local conformationalshape, loops, half-loops, domains, half-domains, sites, termini or anycombination thereof.

As used herein when referring to polynucleotides the term “localconformational shape” means a polynucleotide based structuralmanifestation which is located within a definable region of thepolynucleotide.

As used herein the term “turn” as it relates to nucleic acidconformation means a bend which alters the direction of the backbone ofa polynucleotide and may involve one, two, three or more nucleosideresidues.

As used herein when referring to polynucleotides the term “loop” refersto a structural feature which may serve to reverse the direction of thebackbone of a polynucleotide such that two regions at a distance of thepolynucleotide are brought together spatially. Loops may be open orclosed. Closed loops or “cyclic” loops may include 2, 3, 4, 5, 6, 7, 8,9, 10 or more nucleotides.

As used herein the term “half-loop” refers to a portion of an identifiedloop having at least half the number of nucleic acid resides as the loopfrom which it is derived. It is understood that loops may not alwayscontain an even number nucleic acid residues. Therefore, in those caseswhere a loop contains or is identified to include an odd number, ahalf-loop of the odd-numbered loop will include the whole number portionor next whole number portion of the loop (number of amino acids of theloop/2+/−0.5 amino acids). For example, a loop identified as a7-nucleotide loop could produce half-loops of 3 nucleotides or 4nucleotides (7/2=3.5+/−0.5 being 3 or 4).

As used herein the term “domain” refers to a motif of a polynucleotidehaving one or more identifiable structural or functional characteristicsor properties (e.g., binding capacity, serving as a site forinteractions).

As used herein the term “half-domain” means a portion of an identifieddomain having at least half the number of nucleic acid residues as thedomain from which it is derived. It is understood that domains may notalways contain an even number of nucleic acid residues. Therefore, inthose cases where a domain contains or is identified to include an oddnumber, a half-domain of the odd-numbered domain will include the wholenumber portion or next whole number portion of the domain (number ofnucleotides of the domain/2+/−0.5 nucleotides). For example, a domainidentified as a 7-nucleotide domain could produce half-domains of 3nucleotides or 4 nucleotides (7/2=3.5+/−0.5 being 3 or 4). It is alsounderstood that sub-domains may be identified within domains orhalf-domains, these subdomains possessing less than all of thestructural or functional properties identified in the domains or halfdomains from which they were derived. It is also understood that thenucleotides that include any of the domain types herein need not becontiguous along the backbone of the polynucleotide (i.e., nonadjacentnucleotides may fold structurally to produce a domain, half-domain orsubdomain).

As used herein the terms “site” as it pertains to polynucleotides isused synonymously with “nucleic acid residue” and/or “nucleotide.” Asite represents a position within a polynucleotide that may be modified,manipulated, altered, derivatized or varied.

As used herein the terms “termini” or “terminus” refers to an extremityof a polynucleotide. Such extremity is not limited only to the first orfinal site of the polynucleotide but may include additional nucleotidesin the terminal regions. The polynucleotides of the present disclosuremay be characterized as having both a 5′ and a 3′ terminus.

Once any of the features have been identified or defined as a desiredcomponent of a polynucleotide of the disclosure, any of severalmanipulations and/or modifications of these features may be performed bymoving, swapping, inverting, deleting, randomizing or duplicating.Furthermore, it is understood that manipulation of features may resultin the same outcome as a modification to the molecules of thedisclosure.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to random or site directed mutagenesis.The resulting modified molecules may then be tested for activity usingin vitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present disclosure, the polynucleotides may include aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

In some embodiments, variants of the polynucleotides of the disclosuremay be generated. These variants may have the same or a similar activityas the reference polynucleotide. Alternatively, the variant may have analtered activity (e.g., increased or decreased) relative to a referencepolynucleotide. Generally, variants of a particular polynucleotides ofthe disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% butless than 100% sequence identity to that particular referencepolynucleotide as determined by sequence alignment programs andparameters described herein and known to those skilled in the art. Suchtools for alignment include those of the BLAST suite (Stephen F.Altschul, Thomas L. Madden, Alejandro A. Schiffer, Jinghui Zhang, ZhengZhang. Webb Miller, and David J. Lipman (1997). “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”.Nucleic Acids Res. 25:3389-3402.) Other tools are described herein,specifically in the definition of “identity.”

The engineered polynucleotides of the present disclosure may beincorporated into a vector or plasmid alone or in combination with otherpolynucleotide sequences or features such as those disclosed inInternational Publications WO2007046703 and WO2007148971 (disclosingalternative start codons and AAV vectors produced in insect cells);WO2009104964 (disclosing optimization of expression of AAV proteins ininsect cells and involving alteration of promoter strength, enhancerelements, temperature control); and WO2015137802 (disclosing alternativestart codons, removal of start codons and AAV vectors produced in insectcells), the contents of each of which are incorporated herein byreference in their entirety.

5′ UTR Scaffolds

An embodiment of a 5′ UTR of the present disclosure is presented inFIG. 1. The figure illustrates a promoter 5′ (upstream) of a 5′ UTRwhich includes an “A” region (a 5′ flanking region) which is 5′(upstream) of a hairpin, a “B” region (a 3′ flanking region which caninclude a start codon and kozak nucleotides around the start codon)which is 3′ (downstream) from the stem loop, a “C” region representingthe stem of a stem-loop structure, and a loop (which can range from 4-16nucleotides). In the figure, the Kozak shown is TTT. The promoter and 5′UTR are associated with either a CAP gene (which encodes the structuralcapsid proteins VP1. VP2 and/or VP3) or a REP gene (which encodes thenon-structural replication proteins Rep78 and Rep52).

The engineered 5′ UTRs of the disclosure may have varied G:C content orpercentage. In some embodiments, the 5′ flanking region of the 5′ UTRshave a varied G:C content. In some embodiments, the stem of the 5′ UTRshas a varied G:C content. In some embodiments, the 3′ flanking region ofthe 5′ UTRs has a varied G:C content. In some embodiments, the G:Ccontent of the engineered 5′ UTR is 10-80%, 20-70%, 25%-65%, or 30%-60%.In some embodiments, the G:C content of the engineered 5′ UTR is about25%, about 30%, 34%, about 35%, about 40%, about 45%, about 50%, about55%, 58%, about 60%, 62% or about 65%.

In some embodiments, the engineered 5′ UTR includes or consists ofbetween 80-120 nucleotides, between 90-110 nucleotides, between 95-105nucleotides, between 98-100 nucleotides, or about 99 nucleotides. Insome embodiments, the engineered 5′ UTR includes or consists of 24nucleotides.

In some embodiments, the engineered 5′ UTR is derived from AAV2. In someembodiments, the engineered 5′ UTR is derived from AAV2. In someembodiments, the engineered 5′ UTR is derived from AAVRh10. In someembodiments, the engineered 5′ UTR is derived from AAVPHP.B.

In some embodiments, the engineered 5′ UTR includes or consists ofbetween 98-100 nucleotides, and includes a G:C content of about 25%,about 30%, 34%, about 35%, about 40%, about 45%, about 50%, about 55%,58%, about 60%, 62% or about 65%.

In some embodiments, the engineered 5′ UTR includes a hairpin structure.In some embodiments, the engineered 5′ UTR includes a hairpin structureencoded by a hairpin nucleotide sequence. In some embodiments, thehairpin nucleotide sequence includes a leader sequence. In someembodiments, the hairpin nucleotide sequence includes a leader sequenceand a start codon (e.g. ATG). In some embodiments, the hairpinnucleotide sequence includes a leader sequence, and a start codon (e.g.ATG) within a kozak sequence or modified kozak sequence.

In some embodiments, the engineered 5′ UTR includes a hairpin structurehaving a 5′ flanking region (i.e. upstream region) encoded by a 5′flanking sequence. In some embodiments, the 5′ flanking sequence may beof any length and may be derived in whole or in part from wild type AAVsequence or be completely artificial.

In some embodiments, the engineered 5′ UTR includes a hairpin structurehaving a 3′ flanking region (i.e. downstream region) encoded by a 3′flanking sequence. In some embodiments, the 3′ flanking sequence may beof any length and may be derived in whole or in part from wild type AAVsequence or be completely artificial.

The 5′ flanking sequence and 3′ flanking sequence can have the same sizeand origin, a different size, a different origin, or a different sizeand origin. Either flanking sequence may be absent. The 5′ flankingsequence can include or consist of 2-50, 2-40, 2-30, 2-20, or 2-15nucleotides. The 3′ flanking sequence can include or consist of 2-50,2-40, 2-30, 2-20, or 2-15 nucleotides. The 3′ flanking sequence mayoptionally contain the start codon of an AAV protein or proteins as wellas other sequences such as a Kozak or modified Kozak sequence.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a step-loop structure. In some embodiments, the hairpinstructure includes a stem region and a loop region. In some embodiments,the hairpin structure includes a stem region, a loop region, and astem-complement region. The stem-loop structure can include a stemregion encoded by a stem sequence. The stem-loop structure can include aloop region encoded by a loop sequence. The stem-loop structure caninclude a stem-complement region encoded by a stem-complement sequence.Forming the stem of the stem-loop structure of the hairpin are paired orsubstantially paired nucleobases of between 2 and 50 pairs. The stem maycontain one or more mismatches, bulges or loops. In some embodiments,the stem sequence and the stem-complement sequence are 100%complementary (i.e. zero mismatches). In some embodiments, the stemsequence and the stem-complement sequence include zero, one, two, three,four or five mismatches.

In some embodiments, the engineered 5′ UTR includes a hairpin structurepresented in Table 1. In Table 1, the name includes the parentalserotype (e.g., AAV2), the hairpin number (e.g., HP1 etc.), thecharacteristics of the hairpin are given where the parent serotype isgiven, followed by the length of the hairpin, the number of mismatches(where position is an uppercase letter in the sequence), the location ofthe canonical start codon (e.g., ATG) and the Kozak sequence. Theposition of the loop portion of the hairpin is underlined in thesequence with the canonical ATG start codon in bold. The sequence ofeach hairpin is given in Table 1 as well as the resulting VP1:VP2:VP3incorporation ratio relative to VP3 having a value of 10.

TABLE 1 Ratio (VP1:VP Name Design Characteristic Hairpin Sequence 2:VP3)AAV2- HP1

AAV2 HP: 10 bp 0 mismatch ATG in the loop Kozak: TTTatacgactcgacgaagacttgatc aaccgtcggctttatggct (SEQ ID NO: 1) AAV2- HP2

AAV2 HP: 10 bp 1 mismatch(Base) ATG in the loop Kozak: TTTatacgactcgacgaagacttgatc aaccAtcggctttatggct (SEQ ID NO: 2) AAV2- HP3

AAV2 HP: 10 bp 1 mismatch(loop) ATG in the loop Kozak: TTTatacgactcgacgaagacttgatc aaccgtAggctttatg gct (SEQ ID NO: 3) AAV2- HP4

AAV2 HP: 4 bp 0 mismatch ATG in the loop Kozak: TTTatacgactcgacgaagacttgatc ctgactcggctttatggct (SEQ ID NO: 4) AAV2- HP5

AAV2 HP: 14 bp 1 mismatch ATG in the loop Kozak: TTTatacgactcgacgaagacttagTt aaccgtcggctttatggct (SEQ ID NO: 5) AAV2- HP6

AAV2 HP: 14 bp 2 mismatches ATG in the loop Kozak: TTTatacgactcgacgaagacttagTt aaccgtcCgctttatggct (SEQ ID NO: 6) 34.6  0  10   AAV2- HP7

AAV2 HP: 14 bp 3 mismatches ATG in the loop Kozak: TTTatacgactcgacgaagacttagTt aacTgtcCgctttatggct (SEQ ID NO: 7) 19.1  0  10   AAV2- HP8

AAV2 HP: 5 bp 0 mismatch 3 bp from ATG Kozak: TTTatacgactcgacgaagacctgcca tctaaggcagtttatggct (SEQ ID NO: 8)  2.6  1.310   AAV2- HP9

AAV2 HP: 10 bp 0 mismatch 3 bp from ATG Kozak: TTTatacgactctgccagctcatctaag agctggcagtttatggct (SEQ ID NO: 9)  0.3  0.210   AAV2- HP10

AAV2 HP: 10 bp 1 mismatch 3 bp from ATG Kozak: TTTatacgactctgccTgctcatctaag agctggcagtttatggct (SEQ ID NO: 10)  9.2  0.610   AAV2- atacctgccagctcttcgatctaac HP11 gaagagctggcagtttatggct (SEQ IDNO: 11) AAV2- HP12

AAV2 HP: 14 bp 1 mismatch 3 bp from ATG Kozak: TTTatacctgccTgctcttcgatctaacg aagagctggcagtttatggct (SEQ ID NO: 12)  9.8 0.4 10   AAV2- HP13

AAV2 HP: 14 pb 2 mismatches 3 bp from ATG Kozak: TTTatacctgccTgctcAtcgatctaac gaagagctggcagtttatggct (SEQ ID NO: 13)  7.7 1.0 10   AAV2- HP14

AAV2 HP: 5 bp 0 mismatch 17 bp from ATG Kozak: TTTatacctgccatctaaggcaggactc gacgaagactttatggct (SEQ ID NO: 14) ND AAV2-HP15

AAV2 HP: 14 bp 0 mismatch 17 bp from the ATG Kozak: TTTatacctgccagctcatctaagagct ggcaggacttttatggct (SEQ ID NO: 15) 25.1  0  10   AAV2- HP16

AAV2 HP: 14 bp 1 mismatch 17 pb from ATG Kozak: TTTatacctgccTgctcatctaagagct ggcaggacttttatggct (SEQ ID NO: 16) 17.7  0.310  

In some embodiments, the engineered 5′ UTR includes a hairpin structure,or a component thereof, which is encoded by a hairpin nucleotidesequence selected from SEQ ID NO: 1-16. In some embodiments, theengineered 5′ UTR includes a hairpin structure, or a component thereof,encoded by a nucleotide sequence having at least 60% identity, at least65% identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to SEQ ID NO: 1-16.

In some embodiments, the engineered 5′ UTR includes a Hairpin 1structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a Hairpin 2 structure, or a component thereof. In someembodiments, the engineered 5′ UTR includes a Hairpin 3 structure, or acomponent thereof. In some embodiments, the engineered 5′ UTR includes aHairpin 4 structure, or a component thereof. In some embodiments, theengineered 5′ UTR includes a Hairpin 5 structure, or a componentthereof. In some embodiments, the engineered 5′ UTR includes a Hairpin 6structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a Hairpin 7 structure, or a component thereof. In someembodiments, the engineered 5′ UTR includes a Hairpin 8 structure, or acomponent thereof. In some embodiments, the engineered 5′ UTR includes aHairpin 10 structure, or a component thereof. In some embodiments, theengineered 5′ UTR includes a Hairpin 11 structure, or a componentthereof. In some embodiments, the engineered 5′ UTR includes a Hairpin13 structure, or a component thereof. In some embodiments, theengineered 5′ UTR includes a Hairpin 14 structure, or a componentthereof. In some embodiments, the engineered 5′ UTR includes a Hairpin15 structure, or a component thereof. In some embodiments, theengineered 5′ UTR includes a Hairpin 16 structure, or a componentthereof.

In some embodiments, the engineered 5′ UTR includes a Hairpin 9structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a hairpin structure, or a component thereof, which isencoded by a hairpin nucleotide sequence comprising SEQ ID NO: 9.

In some embodiments, the engineered 5′ UTR includes a Hairpin 12structure, or a component thereof. In some embodiments, the engineered5′ UTR includes a hairpin structure, or a component thereof, which isencoded by a hairpin nucleotide sequence comprising SEQ ID NO: 12.

In some embodiments, the engineered 5′ UTR includes a hairpin structure,or one or more components thereof, selected from the structures andregions presented in Table 2. The disclosure in Table 2 includes: thehairpin number (e.g., HP1) the hairpin sequence (start codon is boldedand loop is underlined) for certain hairpin structures, the upstreamregion sequences (Region A in FIG. 1) for certain hairpin structures,the stem region sequences (Region C in FIG. 1) for certain hairpinstructures, the loop sequences (Region A in FIG. 1) for certain hairpinstructures, the stem complement sequences for certain hairpinstructures, the downstream region sequences (Region B in FIG. 1) forcertain hairpin structures, and the lead sequences for certain hairpinstructures.

TABLE 2 HP Hairpin Upstream Stem Stem Downstream Leader # sequenceRegion (A) Region (C) Loop Complement Region (B) Sequence  1atacgactcgacg atacgactcgacg aaccgtcggc tttatggct Atacgactcgacaagacttgatcaa aagacttgatc SEQ ID NO: 21 gaagacttgatc ccgtcggctttatSEQ ID NO: 17 aaccgtcggctt ggct t SEQ ID NO: 1 SEQ ID NO: 36  2atacgactcgacg atacgactcgacg aaccAtcggc tttatggct atacgactcgacgaagacttgatcaa aagacttgatc SEQ ID NO: 22 aagacttgatcaa ccAtcggctttatSEQ ID NO: 17 ccAtcggcttt ggct SEQ ID NO: 37 SEQ ID NO: 2  3atacgactcgacg atacgactcgacg aaccgtAggc tttatggct atacgactcgacgaagacttgatcaa aagacttgatc SEQ ID NO: 23 aagacttgatcaa ccgtAggctttatSEQ ID NO: 17 ccgtAgcttt ggct SEQ ID NO: 38 SEQ ID NO: 3  4atacgactcgacg atacgactcgacg cggc tttatggct atacgactcgacg aagacttgatcctaagacttgatcct aagacttgatcct gactcggctttat gact gactcggcttt ggctSEQ ID NO: 18 SEQ ID NO: 39 SEQ ID NO: 4  5 atacgactcgacg atacgactcgacgagTtaaccgtcgg tttatggct atacgactcgacg aagacttagTtaa aagactt caagacttagTtaa ccgtcggctttat SEQ ID NO: 19 SEQ ID NO: 24 ccgtcggcttt ggctSEQ ID NO: 40 SEQ ID NO: 5  6 atacgactcgacg atacgactcgacg agTtaaccgtcCgtttatggct atacgactcgacg aagacttagTtaa aagactt c aagacttagTtaaccgtcCgctttat SEQ ID NO: 19 SEQ ID NO: 25 ccgtcCgcttt ggct SEQ ID NO: 41SEQ ID NO: 6  7 atacgactcgacg atacgactcgacg agTtaacTgtcCg tttatggctatacgactcgacg aagacttagTtaa aagactt c aagacttagTtaa cTgtcCgctttatSEQ ID NO: 19 SEQ ID NO: 26 cTgtCgcttt ggct SEQ ID NO: 42 SEQ ID NO: 7 8 atacgactcgacg atacgactcgacg ctgcc atctaa ggcag tttatggctatacgactcgacg aagacctgccatc aagac aagacctgccat taaggcagtttatSEQ ID NO: 20 ctaaggcagttt ggct SEQ ID NO: 43 SEQ ID NO: 8  9atacgactctgcc atacgact ctgccagctc atctaa gagctggcag tttatggctatacgactctgcc agctcatctaaga SEQ ID NO: 27 SEQ ID NO: 32 agctcatctaagagctggcagtttat gctggcagttt ggct SEQ ID NO: 44 SEQ ID NO: 9 10atacgactctgcc atacgact ctgccTgctc atctaa gagctggcag tttatggctatacgactctgcc Tgctcatctaaga SEQ ID NO: 28 SEQ ID NO: 32 Tgctcatctaagagctggcagtttat gctggcagttt ggct SEQ ID NO: 45 SEQ ID NO: 10 11atacctgccagct atac ctgccagctcttc atctaa cgaagagctggca tttatggctatacctgccagct cttcgatctaacg g g cttcgatctaacg aagagctggcag SEQ ID NO: 29SEQ ID NO: 33 aagagctggcagt tttatggct tt SEQ ID NO: 11 SEQ ID NO: 46 12atacctgccTgct atac ctgccTgctcttc atctaa cgaagagctggca tttatggctatacctgccTgct cttcgatctaacg g g cttcgatctaacg aagagctggcag SEQ ID NO: 30SEQ ID NO: 33 aagagctggcagt tttatggct tt SEQ ID NO: 12 SEQ ID NO: 47 13atacctgccTgct atac ctgccTgctcAtc atctaa cgaagagctggca tttatggctatacctgccTgct cAtcgatctaacg g g cAtcgatctaacg aagagctggcagtSEQ ID NO: 31 SEQ ID NO: 33 aagagctggcagt ttatggct tt SEQ ID NO: 13SEQ ID NO: 48 14 atacctgccatct atac ctgcc atctaa ggcag gactcgacgaagaatacctgccatct aaggcaggactcg ctttatggct aaggcaggactcg acgaagactttatSEQ ID NO: 34 acgaagacttt ggct SEQ ID NO: 49 SEQ ID NO: 14 15atacctgccagct atac ctgccagctc atctaa gagctggcag gacttttatggctatacctgccagct catctaagagctg SEQ ID NO: 27 SEQ ID NO: 32 SEQ ID NO: 35catctaagagctg gcaggacttttat gcaggactttt ggct SEQ ID NO: 50 SEQ ID NO: 1516 atacctgccTgct atac ctgccTgctc atctaa gagctggcag gacttttatggctatacctgccTgct catctaagagctg SEQ ID NO: 28 SEQ ID NO: 32 SEQ ID NO: 35catctaagagctg gcaggacttttat gcaggactttt ggct SEQ ID NO: 51 SEQ ID NO: 16

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes an upstream region encoded by a nucleotide sequenceselected from atacgact, atac, and SEQ ID NO: 17-20. In some embodiments,the engineered 5′ UTR includes a hairpin structure which includes anupstream region encoded by a nucleotide sequence having at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity or at least 95% identity to a nucleotide sequence selected fromatacgact, atac, and SEQ ID NO: 17-20. The upstream region may be of anylength, including (but not limited to) between 4-30 nucleotides, between4-20 nucleotides, between 4-15 nucleotides, between 5-15 nucleotides,between 6-12 nucleotides, 6 nucleotides, 7, nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides or 20 nucleotides.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a downstream region encoded by a nucleotide sequenceselected from tttatggct, attatggct, tatatggct, ttaatggct, taaatggct,ataatggct, and SEQ ID NO: 34-35. In some embodiments, the engineered 5′UTR includes a hairpin structure which includes an downstream regionencoded by a nucleotide sequence having at least 60% identity, at least65% identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from tttatggct, attatggct,tatatggct, ttaatggct, taaatggct, ataatggct, and SEQ ID NO: 34-35. Theupstream region may be of any length, including (but not limited to)between 0-30 nucleotides, between 0-20 nucleotides, between 0-15nucleotides, between 4-15 nucleotides, between 6-12 nucleotides, 0nucleotides, 1, nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides,5, nucleotides, 6 nucleotides, 7, nucleotides, 8 nucleotides, 9nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides or 20 nucleotides.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a stem region encoded by a nucleotide sequence selectedfrom cggc, ctgcc and SEQ ID NO: 21-31. In some embodiments, theengineered 5′ UTR includes a hairpin structure which includes an stemregion encoded by a nucleotide sequence having at least 60% identity, atleast 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity or atleast 95% identity to a nucleotide sequence selected from cggc, ctgccand SEQ ID NO: 21-31. The stem region may be of any length, including(but not limited to) between 4-30 nucleotides, between 4-20 nucleotides,between 4-15 nucleotides, between 5-15 nucleotides, between 6-12nucleotides, 4 nucleotides, 5, nucleotides, 6 nucleotides, 7,nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides or 20 nucleotides.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a stem complement region encoded by a nucleotide sequenceselected from ggcag and SEQ ID NO: 32-33. In some embodiments, theengineered 5′ UTR includes a hairpin structure which includes an stemcomplement region encoded by a nucleotide sequence having at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity or at least 95% identity to a nucleotide sequence selected fromggcag and SEQ ID NO: 32-33. The stem complement region may be of anylength, including (but not limited to) between 0-30 nucleotides, between0-20 nucleotides, between 0-15 nucleotides, between 4-15 nucleotides,between 6-12 nucleotides, 0 nucleotides, 1, nucleotide, 2 nucleotides, 3nucleotides, 4 nucleotides, 5, nucleotides, 6 nucleotides, 7,nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides or 20 nucleotides.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a loop region encoded by a nucleotide sequence selectedfrom tttatggct and atctaa. In some embodiments, the engineered 5′ UTRincludes a hairpin structure which includes a loop region encoded by anucleotide sequence having at least 60% identity, at least 65% identity,at least 70% identity, at least 75% identity, at least 80% identity, atleast 85% identity, at least 90;% identity or at least 95% identity to anucleotide sequence selected from tttatggct and atctaa. The upstreamregion may be of any length, including (but not limited to) between 4-30nucleotides, between 4-20 nucleotides, between 4-15 nucleotides, between5-15 nucleotides, between 6-12 nucleotides, 4, nucleotides, 5nucleotides, 6 nucleotides, 7, nucleotides, 8 nucleotides, 9nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides or 20 nucleotides.

In some embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a leader sequence selected from SEQ ID NO: 36-51. In someembodiments, the engineered 5′ UTR includes a hairpin structure whichincludes a leader sequence selected from SEQ ID NO: 59-63. In someembodiments, the engineered 5′ UTR includes a hairpin structure whichincludes a leader sequence having at least 60% identity, at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from SEQ ID NO: 36-51. Insome embodiments, the engineered 5′ UTR includes a hairpin structurewhich includes a leader sequence having at least 60% identity, at least65% identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from SEQ ID NO: 59-63.

As used herein a “5′ UTR scaffold” is a framework or startingpolynucleotide that forms the sequence or structural basis against whichto design or make a subsequent engineered polynucleotide.

In some embodiments, an engineering 5′ UTR is engineered according toone or more of the following parameters: length of 5′ flanking region,length of 5′ stem arm, length of loop, length of 3′ stem arm, length of3′ flanking region. G:C % in hairpin sequence, and the number of stemmismatches.

The 5′ and 3′ arms of the stem may be completely complementary across asubstantial portion of their length. In other embodiments, the 5′ and 3′arms of the stem may be at least 70, 80, 90, 95 or 99% complementaryacross independently at least 50, 60, 70, 80, 85, 90, 95, or 99% of thelength.

Separating the 5′ and 3′ arms of the stem of the stem loop structure isa loop (also known as a loop motif). The loop may be of any length,between 4-30 nucleotides, between 4-20 nucleotides, between 4-15nucleotides, between 5-15 nucleotides, between 6-12 nucleotides, 6nucleotides, 7, nucleotides, 8 nucleotides, 9 nucleotides, 10nucleotides, 11 nucleotides, and/or 12 nucleotides.

In some embodiments, the loop includes the start codon of the AAVprotein.

Spacer regions may be present in the engineered 5′ UTRs to separate oneor more features from one another. There may be one or more such spacerregions present.

In some embodiments, a spacer region of between 8-20. i.e., 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be presentbetween any two or more features.

In some embodiments, the engineered 5′ UTR includes in the 5′ to 3′direction, a 5′ flanking sequence, a 5′ arm, a loop motif, a 3′ arm anda 3′ flanking sequence.

In some embodiments, the 5′ arm, loop motif and/or 3′ arm sequence maybe altered (e.g., substituting 1 or more nucleotides, adding nucleotidesand/or deleting nucleotides). The alteration may cause a beneficialchange in the function of the construct.

In some embodiments, the modulatory polynucleotide is designed using atleast one of the following properties: loop variant,mismatch/bulge/wobble variant, stem mismatch, loop variant, stemmismatch variant or a stem sequence variant.

One embodiment of a viral expression construct of the present disclosureis shown in FIG. 2A. The expression control sequence of the viralexpression construct includes a p10 promoter (SEQ ID NO: 52) and anengineered 5′ UTR which includes an ATG start codon within AAV2-HP9 (SEQID NO: 9). The viral expression construct also includes an AAV2-CAPsequence coding for VP proteins (SEQ ID NO: 53). The viral expressionconstruct is encoded by SEQ ID NO: 54.

One embodiment of a viral expression construct of the present disclosureis shown in FIG. 2B. The expression control sequence of the viralexpression construct includes a polH promoter (SEQ ID NO: 55) and anengineered 5′ UTR which includes an ATG start codon within AAV2-HP9 (SEQID NO: 9) or a modified AAV2-HP9 (SEQ ID NO: 56). The viral expressionconstruct also includes a REP sequence coding for Rep proteins (SEQ IDNO: 57). The viral expression construct is encoded by SEQ ID NO: 58.

One embodiment of a viral expression construct of the present disclosureis shown in FIG. 2C, which includes both the CAP viral expressionconstruct from FIG. 2A (SEQ ID NO: 54) and the REP viral expressionconstruct from FIG. 2B (SEQ ID NO: 58).

Viral Production Cells and Vectors Mammalian-Production System

Viral production of the present disclosure disclosed herein describesprocesses and methods for producing AAV particles or viral vector thatcontacts a target cell to deliver a payload construct, e.g. arecombinant AAV particle or viral construct, which includes a nucleotideencoding a payload molecule. The viral production cell may be selectedfrom any biological organism, including prokaryotic (e.g., bacterial)cells, and eukaryotic cells, including, insect cells, yeast cells andmammalian cells.

In certain embodiments, the AAV particles of the present disclosure maybe produced in a viral production cell that includes a mammalian cell.Viral production cells may comprise mammalian cells such as A549, WEH1,3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO. W138,HeLa, HEK293, HEK293T (293T), Saos, C2C12, L cells, HT1080. HepG2 andprimary fibroblast, hepatocyte and myoblast cells derived from mammals.Viral production cells can include cells derived from mammalian speciesincluding, but not limited to, human, monkey, mouse, rat, rabbit, andhamster or cell type, including but not limited to fibroblast,hepatocyte, tumor cell, cell line transformed cell, etc.

AAV viral production cells commonly used for production of recombinantAAV particles include, but is not limited to HEK293 cells, COS cells,C127, 3T3, CHO, HeLa cells, KB cells, BHK, and other mammalian celllines as described in U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741,683,5,691.176, 6,428,988 and 5,688,676: U.S. patent application2002/0081721, and International Patent Publication Nos. WO 00/47757. WO00/24916, and WO 96/17947, the contents of each of which are hereinincorporated by reference in their entireties. In certain embodiments,the AAV viral production cells are trans-complementing packaging celllines that provide functions deleted from a replication-defective helpervirus, e.g., HEK293 cells or other Ea trans-complementing cells.

In certain embodiments, the packaging cell line 293-10-3 (ATCC AccessionNo. PTA-2361) may be used to produce the AAV particles, as described inU.S. Pat. No. 6,281,010, the contents of which are herein incorporatedby reference in its entirety.

In certain embodiments, of the present disclosure a cell line, such as aHeLA cell line, for trans-complementing E1 deleted adenoviral vectors,which encoding adenovirus Ela and adenovirus E1b under the control of aphosphoglycerate kinase (PGK) promoter can be used for AAV particleproduction as described in U.S. Pat. No. 6,365,394, the contents ofwhich are incorporated herein by reference in their entirety.

In certain embodiments, AAV particles are produced in mammalian cellsusing a triple transfection method wherein a payload construct,parvoviral Rep and parvoviral Cap and a helper construct are comprisedwithin three different constructs. The triple transfection method of thethree components of AAV particle production may be utilized to producesmall lots of virus for assays including transduction efficiency, targettissue (tropism) evaluation, and stability.

AAV particles to be formulated may be produced by triple transfection orbaculovirus mediated virus production, or any other method known in theart. Any suitable permissive or packaging cell known in the art may beemployed to produce the vectors. In certain embodiments,trans-complementing packaging cell lines are used that provide functionsdeleted from a replication-defective helper virus, e.g., 293 cells orother Ela trans-complementing cells.

The gene cassette may contain some or all of the parvovirus (e.g., AAV)cap and rep genes. In certain embodiments, some or all of the cap andrep functions are provided in trans by introducing a packaging vector(s)encoding the capsid and/or Rep proteins into the cell. In certainembodiments, the gene cassette does not encode the capsid or Repproteins. Alternatively, a packaging cell line is used that is stablytransformed to express the cap and/or rep genes.

Recombinant AAV virus particles are, in certain embodiments, producedand purified from culture supernatants according to the procedure asdescribed in US2016/0032254, the contents of which are incorporated byreference. Production may also involve methods known in the artincluding those using 293T cells, triple transfection or any suitableproduction method.

In certain embodiments, mammalian viral production cells (e.g 293Tcells) can be in an adhesion/adherent state (e.g. with calciumphosphate) or a suspension state (e.g with polyethylenimine (PEI)). Themammalian viral production cell is transfected with plasmids requiredfor production of AAV, (i.e., AAV rep/cap construct, an adenoviralhelper construct, and/or ITR flanked payload construct). In certainembodiments, the transfection process can include optional mediumchanges (e.g. medium changes for cells in adhesion form, no mediumchanges for cells in suspension form, medium changes for cells insuspension form if desired). In certain embodiments, the transfectionprocess can include transfection mediums such as DMEM or F17. In certainembodiments, the transfection medium can include serum or can beserum-free (e.g. cells in adhesion state with calcium phosphate and withserum, cells in suspension state with PEI and without serum).

Cells can subsequently be collected by scraping (adherent form) and/orpelleting (suspension form and scraped adherent form) and transferredinto a receptacle. Collection steps can be repeated as necessary forfull collection of produced cells. Next, cell lysis can be achieved byconsecutive freeze-thaw cycles (−80 C to 37 C), chemical lysis (such asadding detergent triton), mechanical lysis, or by allowing the cellculture to degrade after reaching ˜0% viability. Cellular debris isremoved by centrifugation and/or depth filtration. The samples arequantified for AAV particles by DNase resistant genome titration by DNAqPCR.

AAV particle titers are measured according to genome copy number (genomeparticles per milliliter). Genome particle concentrations are based onDNA qPCR of the vector DNA as previously reported (Clark et al. (1999)Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther.,6:272-278).

Insect Cells

Viral production of the present disclosure includes processes andmethods for producing AAV particles or viral vectors that contact atarget cell to deliver a payload construct, e.g. a recombinant viralconstruct, which includes a nucleotide encoding a payload molecule. Incertain embodiments, the AAV particles or viral vectors of the presentdisclosure may be produced in a viral production cell that includes aninsect cell.

Growing conditions for insect cells in culture, and production ofheterologous products in insect cells in culture are well-known in theart, see U.S. Pat. No. 6,204,059, the contents of which are hereinincorporated by reference in their entirety.

Any insect cell which allows for replication of parvovirus and which canbe maintained in culture can be used in accordance with the presentdisclosure. AAV viral production cells commonly used for production ofrecombinant AAV particles include, but is not limited to, Spodopterafrugiperda, including, but not limited to the Sf9 or Sf21 cell lines,Drosophila cell lines, or mosquito cell lines, such as Aedes albopictusderived cell lines. Use of insect cells for expression of heterologousproteins is well documented, as are methods of introducing nucleicacids, such as vectors, e.g., insect-cell compatible vectors, into suchcells and methods of maintaining such cells in culture. See, forexample, Methods In Molecular Biology, ed. Richard, Humana Press, NJ(1995): O'Reilly et al., Baculovirus Expression Vectors, A LaboratoryManual, Oxford Univ. Press (1994): Samulski et al., J. Vir. 63:3822-8(1989): Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991);Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir.219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); and Samulski etal., U.S. Pat. No. 6,204,059, the contents of each of which are hereinincorporated by reference in their entirety.

In one embodiment, the AAV particles are made using the methodsdescribed in WO2015/191508, the contents of which are hereinincorporated by reference in their entirety.

In certain embodiments, insect host cell systems, in combination withbaculoviral systems (e.g., as described by Luckow et al., Bio/Technology6: 47 (1988)) may be used. In certain embodiments, an expression systemfor preparing chimeric peptide is Trichoplusia ni, Tn 5B1-4 insectcells/baculoviral system, which can be used for high levels of proteins,as described in U.S. Pat. No. 6,660,521, the contents of which areherein incorporated by reference in their entirety.

Expansion, culturing, transfection, infection and storage of insectcells can be carried out in any cell culture media, cell transfectionmedia or storage media known in the art, including Hyclone SFX InsectCell Culture Media, Expression System ESF AF Insect Cell Culture Medium,ThermoFisher Sf900II media, ThermoFisher Sf900III media, or ThermoFisherGrace's Insect Media. Insect cell mixtures of the present disclosure canalso include any of the formulation additives or elements described inthe present disclosure, including (but not limited to) salts, acids,bases, buffers, surfactants (such as Poloxamer 188/Pluronic F-68), andother known culture media elements. Formulation additives can beincorporated gradually or as “spikes” (incorporation of large volumes ina short time).

Baculovirus-Production System

In certain embodiments, processes of the present disclosure can includeproduction of AAV particles or viral vectors in a baculoviral systemusing a viral expression construct and a payload construct vector. Incertain embodiments, the baculoviral system includes Baculovirusexpression vectors (BEVs) and/or baculovirus infected insect cells(BIICs). In certain embodiments, a viral expression construct or apayload construct of the present disclosure can be a bacmid, also knownas a baculovirus plasmid. In certain embodiments, a viral expressionconstruct or a payload construct of the present disclosure can bepolynucleotide incorporated by homologous recombination (transposondonor/acceptor system) into a bacmid by standard molecular biologytechniques known and performed by a person skilled in the art.Transfection of separate viral replication cell populations produces twoor more groups (e.g. two, three) of baculoviruses (BEVs), one or moregroup that includes the viral expression construct (Expression BEV), andone or more group that includes the payload construct (Payload BEV). Thebaculoviruses may be used to infect a viral production cell forproduction of AAV particles or viral vector.

In certain embodiments, the process includes transfection of a singleviral replication cell population to produce a single baculovirus (BEV)group which includes both the viral expression construct and the payloadconstruct. These baculoviruses may be used to infect a viral productioncell for production of AAV particles or viral vector.

In certain embodiments, BEVs are produced using a Bacmid Transfectionagent, such as Promega FuGENE HD, WFI water, or hermoFisher CellfectinII Reagent. In certain embodiments, BEVs are produced and expanded inviral production cells, such as an insect cell.

In certain embodiments, the method utilizes seed cultures of viralproduction cells that include one or more BEVs, including baculovirusinfected insect cells (BIICs). The seed BIICs have beentransfected/transduced/infected with an Expression BEV which includes aviral expression construct, and also a Payload BEV which includes apayload construct. In certain embodiments, the seed cultures areharvested, divided into aliquots and frozen, and may be used at a latertime to initiate transfection/transduction/infection of a naïvepopulation of production cells. In certain embodiments, a bank of seedBIICs is stored at −80° C. or in LN₂ vapor.

Baculoviruses are made of several essential proteins which are essentialfor the function and replication of the Baculovirus, such as replicationproteins, envelope proteins and capsid proteins. The Baculovirus genomethus includes several essential-gene nucleotide sequences encoding theessential proteins. As a non-limiting example, the genome can include anessential-gene region which includes an essential-gene nucleotidesequence encoding an essential protein for the Baculovirus construct.The essential protein can include: GP64 baculovirus envelope protein,VP39 baculovirus capsid protein, or other similar essential proteins forthe Baculovirus construct.

Baculovirus expression vectors (BEV) for producing AAV particles ininsect cells, including but not limited to Spodoptera frugiperda (Sf9)cells, provide high titers of viral vector product. Recombinantbaculovirus encoding the viral expression construct and payloadconstruct initiates a productive infection of viral vector replicatingcells. Infectious baculovirus particles released from the primaryinfection secondarily infect additional cells in the culture,exponentially infecting the entire cell culture population in a numberof infection cycles that is a function of the initial multiplicity ofinfection, see Urabe, M. et al. J Virol. 2006 February:80(4):1874-85,the contents of which are herein incorporated by reference in theirentirety.

Production of AAV particles with baculovirus in an insect cell systemmay address known baculovirus genetic and physical instability.

In certain embodiments, the production system of the present disclosureaddresses baculovirus instability over multiple passages by utilizing atiterless infected-cells preservation and scale-up system. Small scaleseed cultures of viral producing cells are transfected with viralexpression constructs encoding the structural and/or non-structuralcomponents of the AAV particles. Baculovirus-infected viral producingcells are harvested into aliquots that may be cryopreserved in liquidnitrogen; the aliquots retain viability and infectivity for infection oflarge scale viral producing cell culture Wasilko D J et al. Protein ExprPurif. 2009 June; 65(2):122-32, the contents of which are hereinincorporated by reference in their entirety.

A genetically stable baculovirus may be used to produce a source of theone or more of the components for producing AAV particles ininvertebrate cells. In certain embodiments, defective baculovirusexpression vectors may be maintained episomally in insect cells. In suchan embodiment the bacmid vector is engineered with replication controlelements, including but not limited to promoters, enhancers, and/orcell-cycle regulated replication elements.

In certain embodiments, baculoviruses may be engineered with a (non-)selectable marker for recombination into the chitinase/cathepsin locus.The chia/v-cath locus is non-essential for propagating baculovirus intissue culture, and the V-cath (EC 3.4.22.50) is a cysteine endoproteasethat is most active on Arg-Arg dipeptide containing substrates. TheArg-Arg dipeptide is present in densovirus and parvovirus capsidstructural proteins but infrequently occurs in dependovirus VP1.

In certain embodiments, stable viral producing cells permissive forbaculovirus infection are engineered with at least one stable integratedcopy of any of the elements necessary for AAV replication and vectorproduction including, but not limited to, the entire AAV genome, Rep andCap genes, Rep genes, Cap genes, each Rep protein as a separatetranscription cassette, each VP protein as a separate transcriptioncassette, the AAP (assembly activation protein), or at least one of thebaculovirus helper genes with native or non-native promoters.

In certain embodiments, the Baculovirus expression vectors (BEV) arebased on the AcMNPV baculovirus or BmNPV baculovirus BmNPV.

In certain embodiments, the Baculovirus expression vectors (BEV) is aBEV in which the baculoviral v-cath gene has been deleted (“v-cathdeleted BEV”) or mutated.

Other

In certain embodiments expression hosts include, but are not limited to,bacterial species within the genera Escherichia, Bacillus, Pseudomonas.Salmonella.

In certain embodiments, a host cell which includes AAV rep and cap genesstably integrated within the cell's chromosomes, may be used for AAVparticle production. In a non-limiting example, a host cell which hasstably integrated in its chromosome at least two copies of an AAV repgene and AAV cap gene may be used to produce the AAV particle accordingto the methods and constructs described in U.S. Pat. No. 7,238,526, thecontents of which are incorporated herein by reference in theirentirety.

In certain embodiments, the AAV particle can be produced in a host cellstably transformed with a molecule comprising the nucleic acid sequenceswhich permit the regulated expression of a rare restriction enzyme inthe host cell, as described in US20030092161 and EP 1183380, thecontents of which are herein incorporated by reference in theirentirety.

In certain embodiments, production methods and cell lines to produce theAAV particle may include, but are not limited to those taught inPCT/US1996/010245, PCT/US1997/015716, PCT/US1997/015691.PCT/US1998/019479, PCT/US1998/019463, PCT/US2000/000415,PCT/US2000/040872, PCT/US2004/016614, PCT/US2007/010055,PCT/US1999/005870, PCT/US2000/004755, U.S. Ser. No. 08/549,489, U.S.Ser. No. 08/462,014, US09/659203, U.S. Ser. No. 10/246,447, U.S. Ser.No. 10/465,302, U.S. Pat. Nos. 6,281,010, 6,270,996, 6,261,551,5,756,283 (Assigned to NIH), U.S. Pat. Nos. 6,428,988, 6,274,354,6,943,019, 6,482,634, (Assigned to NIH: U.S. Pat. Nos. 7,238,526,6,475,769), 6,365,394 (Assigned to NIH), U.S. Pat. Nos. 7,491,508,7,291,498, 7,022,519, 6,485,966, 6,953,690, 6,258,595, EP2018421,EP1064393, EP1163354. EP835321, EP931158, EP950111. EP1015619,EP1183380, EP2018421, EP1226264, EP1636370, EP1163354, EP1064393,US20030032613, US20020102714, US20030073232, US20030040101 (Assigned toNIH), US20060003451, US20020090717, US20030092161, US20070231303, U.S.Pat. No. 2,006,0211115, US20090275107, US2007004042. US20030119191,US20020019050, the contents of each of which are incorporated herein byreference in their entirety.

III. Definitions

At various places in the present disclosure, substituents or propertiesof compounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual or subcombination of the members of suchgroups and ranges.

Unless stated otherwise, the following terms and phrases have themeanings described below. The definitions are not meant to be limitingin nature and serve to provide a clearer understanding of certainaspects of the present disclosure.

About: As used herein, the term “about” means+/−10% of the recitedvalue.

Adeno-associated virus: The term “adeno-associated virus” or “AAV” asused herein refers to members of the dependovirus genus comprising anyparticle, sequence, gene, protein, or component derived therefrom.

AAV Particle: As used herein, an “AAV particle” is a virus whichincludes a capsid and a viral genome with at least one payload regionand at least one ITR region. AAV particles of the present disclosure maybe produced recombinantly and may be based on adeno-associated virus(AAV) parent or reference sequences. AAV particle may be derived fromany serotype, described herein or known in the art, includingcombinations of serotypes (i.e., “pseudotyped” AAV) or from variousgenomes (e.g., single stranded or self-complementary). In addition, theAAV particle may be replication defective and/or targeted.

Activity: As used herein, the term “activity” refers to the condition inwhich things are happening or being done. Compositions of the presentdisclosure may have activity and this activity may involve one or morebiological events.

Administering: As used herein, the term “administering” refers toproviding a pharmaceutical agent or composition to a subject.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In certain embodiments, they are administered within about 60,30, 15, 10, 5, or 1 minute of one another. In certain embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Amelioration: As used herein, the term “amelioration” or “ameliorating”refers to a lessening of severity of at least one indicator of acondition or disease. For example, in the context of neurodegenerationdisorder, amelioration includes the reduction of neuron loss.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In certain embodiments, “animal” refers to humans at anystage of development. In certain embodiments, “animal” refers tonon-human animals at any stage of development. In certain embodiments,the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, arabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).In certain embodiments, animals include, but are not limited to,mammals, birds, reptiles, amphibians, fish, and worms. In certainembodiments, the animal is a transgenic animal, genetically-engineeredanimal, or a clone.

Antisense strand: As used herein, the term “the antisense strand” or“the first strand” or “the guide strand” of a siRNA molecule refers to astrand that is substantially complementary to a section of about 10-50nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of themRNA of the gene targeted for silencing. The antisense strand or firststrand has sequence sufficiently complementary to the desired targetmRNA sequence to direct target-specific silencing, e.g., complementaritysufficient to trigger the destruction of the desired target mRNA by theRNAi machinery or process.

Approximately: As used herein, the term “approximately” or “about.” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” refers to a range of values that fall within 25%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or less than)of the stated reference value unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% ofa possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization-based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Baculoviral expression vector (BEV): As used herein a BEV is abaculoviral expression vector, i.e., a polynucleotide vector ofbaculoviral origin. Systems using BEVs are known as baculoviralexpression vector systems (BEVSs).

mBEV or modified BEV: As used herein, a modified BEV is an expressionvector of baculoviral origin which has been altered from a starting BEV(whether wild type or artificial) by the addition and/or deletion and/orduplication and/or inversion of one or more: genes; gene fragments;cleavage sites; restriction sites; sequence regions; sequence(s)encoding a payload or gene of interest; or combinations of theforegoing.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may affect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent.

BIIC: As used herein a BIIC is a baculoviral infected insect cell.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, anAAV particle of the present disclosure may be considered biologicallyactive if even a portion of the encoded payload is biologically activeor mimics an activity considered biologically relevant.

Capsid: As used herein, the term “capsid” refers to the protein shell ofa virus particle.

Codon optimized: As used herein, the terms “codon optimized” or “codonoptimization” refers to a modified nucleic acid sequence which encodesthe same amino acid sequence as a parent/reference sequence, but whichhas been altered such that the codons of the modified nucleic acidsequence are optimized or improved for expression in a particular system(such as a particular species or group of species). As a non-limitingexample, a nucleic acid sequence which includes an AAV capsid proteincan be codon optimized for expression in insect cells or in a particularinsect cell such Spodoptera frugiperda cells. Codon optimization can becompleted using methods and databases known to those in the art.

Complementary and substantially complementary: As used herein, the term“complementary” refers to the ability of polynucleotides to form basepairs with one another. Base pairs are typically formed by hydrogenbonds between nucleotide units in antiparallel polynucleotide strands.Complementary polynucleotide strands can form base pair in theWatson-Crick manner (e.g., A to T, A to U. C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenosine.However, when a U is denoted in the context of the present disclosure,the ability to substitute a T is implied, unless otherwise stated.Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can formhydrogen bond with a nucleotide unit of a second polynucleotide strand.Less than perfect complementarity refers to the situation in which some,but not all, nucleotide units of two strands can form hydrogen bond witheach other. For example, for two 20-mers, if only two base pairs on eachstrand can form hydrogen bond with each other, the polynucleotidestrands exhibit 10% complementarity. In the same example, if 18 basepairs on each strand can form hydrogen bonds with each other, thepolynucleotide strands exhibit 90% complementarity. As used herein, theterm “substantially complementary” means that the siRNA has a sequence(e.g., in the antisense strand) which is sufficient to bind the desiredtarget mRNA, and to trigger the RNA silencing of the target mRNA.

Compound: Compounds of the present disclosure include all of theisotopes of the atoms occurring in the intermediate or final compounds.“Isotopes” refers to atoms having the same atomic number but differentmass numbers resulting from a different number of neutrons in thenuclei. For example, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conditionally active: As used herein, the term “conditionally active”refers to a mutant or variant of a wild-type polypeptide, wherein themutant or variant is more or less active at physiological conditionsthan the parent polypeptide. Further, the conditionally activepolypeptide may have increased or decreased activity at aberrantconditions as compared to the parent polypeptide. A conditionally activepolypeptide may be reversibly or irreversibly inactivated at normalphysiological conditions or aberrant conditions.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In certain embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In certainembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In certainembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In certainembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In certain embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an polynucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Control Elements: As used herein, “control elements”. “regulatorycontrol elements” or “regulatory sequences” refers to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which provide for thereplication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always be presentas long as the selected coding sequence is capable of being replicated,transcribed and/or translated in an appropriate host cell.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering an AAV particle, a compound, substance, entity, moiety, cargoor payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of an AAVparticle to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dosing regimen: As used herein, a “dosing regimen” is a schedule ofadministration or physician determined regimen of treatment,prophylaxis, or palliative care.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Engineered: As used herein, embodiments of the present disclosure are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats cancer, an effectiveamount of an agent is, for example, an amount sufficient to achievetreatment, as defined herein, of cancer, as compared to the responseobtained without administration of the agent.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription): (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least one AAVparticle and a delivery agent or excipient.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may include polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Gene expression: The term “gene expression” refers to the process bywhich a nucleic acid sequence undergoes successful transcription and inmost instances translation to produce a protein or peptide. For clarity,when reference is made to measurement of “gene expression”, this shouldbe understood to mean that measurements may be of the nucleic acidproduct of transcription, e.g., RNA or mRNA or of the amino acid productof translation, e.g., polypeptides or peptides. Methods of measuring theamount or levels of RNA, mRNA, polypeptides and peptides are well knownin the art.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In certain embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the present disclosure, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In certain embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the present disclosure, two protein sequences areconsidered to be homologous if the proteins are at least about 50%, 60%,70%, 80%, or 90% identical for at least one stretch of at least about 20amino acids.

Heterologous Region: As used herein the term “heterologous region”refers to a region which would not be considered a homologous region.

Homologous Region: As used herein the term “homologous region” refers toa region which is similar in position, structure, evolution origin,character, form or function.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology. Lesk, A. M., ed., Oxford UniversityPress. New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993: Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987: ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer.Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988): incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to. GCG programpackage, Devereux. J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In certain embodiments, isolated agents aremore than about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components.

Substantially isolated: By “substantially isolated” is meant that asubstance is substantially separated from the environment in which itwas formed or detected. Partial separation can include, for example, acomposition enriched in the substance or AAV particles of the presentdisclosure. Substantial separation can include compositions containingat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,or at least about 99% by weight of the compound of the presentdisclosure, or salt thereof. Methods for isolating compounds and theirsalts are routine in the art.

Linker: As used herein “linker” refers to a molecule or group ofmolecules which connects two molecules. A linker may be a nucleic acidsequence connecting two nucleic acid sequences encoding two differentpolypeptides. The linker may or may not be translated. The linker may bea cleavable linker.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the present disclosure. Molecules may bemodified in many ways including chemically, structurally, andfunctionally. As used herein, embodiments of the disclosure are“modified” when they have or possess a feature or property, whetherstructural or chemical, that varies from a starting point, wild type ornative molecule.

Mutation: As used herein, the term “mutation” refers to any changing ofthe structure of a gene, resulting in a variant (also called “mutant”)form that may be transmitted to subsequent generations. Mutations in agene may be caused by the alternation of single base in DNA, or thedeletion, insertion, or rearrangement of larger sections of genes orchromosomes.

Naturally Occurring: As used herein, “naturally occurring” or“wild-type” means existing in nature without artificial aid, orinvolvement of the hand of man.

Non-human vertebrate: As used herein, a “non-human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon within the givenreading frame, other than at the end of the reading frame.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Payload: As used herein, “payload” or “payload region” refers to one ormore polynucleotides or polynucleotide regions encoded by or within aviral genome or an expression product of such polynucleotide orpolynucleotide region. e.g., a transgene, a polynucleotide encoding apolypeptide or multi-polypeptide, or a modulatory nucleic acid orregulatory nucleic acid.

Payload construct: As used herein, “payload construct” is one or morevector production construct which includes a polynucleotide regionencoding or comprising a payload that is flanked on one or both sides byan inverted terminal repeat (ITR) sequence. The payload constructpresents a template that is replicated in a viral production cell toproduce a therapeutic viral genome within a viral particle.

Payload construct vector: As used herein, “payload construct vector” isa vector encoding or comprising a payload construct, and regulatoryregions for replication and expression of the payload construct inbacterial cells.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, acetic acid,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile can be used. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties,Selection. and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH,2008, and Berge et al., Journal of Pharmaceutical Science. 66, 1-19(1977), each of which is incorporated herein by reference in itsentirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the presentdisclosure wherein molecules of a suitable solvent are incorporated inthe crystal lattice. A suitable solvent is physiologically tolerable atthe dosage administered. For example, solvates may be prepared bycrystallization, recrystallization, or precipitation from a solutionthat includes organic solvents, water, or a mixture thereof. Examples ofsuitable solvents are ethanol, water (for example, mono-, di-, andtri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body: (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” or “prevention” refersto partially or completely delaying onset of an infection, disease,disorder and/or condition; partially or completely delaying onset of oneor more symptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic orcourse of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure takento maintain health and prevent the spread of disease.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection. “Purified” refers to the state ofbeing pure. “Purification” refers to the process of making pure.

Region: As used herein, the term “region” refers to a zone or generalarea. In certain embodiments, when referring to a protein or proteinmodule, a region may include a linear sequence of amino acids along theprotein or protein module or may include a three-dimensional area, anepitope and/or a cluster of epitopes. In certain embodiments, regionsinclude terminal regions. As used herein, the term “terminal region”refers to regions located at the ends or termini of a given agent. Whenreferring to proteins, terminal regions may include N- and/or C-termini.N-termini refer to the end of a protein comprising an amino acid with afree amino group. C-termini refer to the end of a protein comprising anamino acid with a free carboxyl group. N- and/or C-terminal regions maythere for include the N- and/or C-termini as well as surrounding aminoacids. In certain embodiments, N- and/or C-terminal regions include fromabout 3 amino acid to about 30 amino acids, from about 5 amino acids toabout 40 amino acids, from about 10 amino acids to about 50 amino acids,from about 20 amino acids to about 100 amino acids and/or at least 100amino acids. In certain embodiments, N-terminal regions may include anylength of amino acids that includes the N-terminus but does not includethe C-terminus. In certain embodiments, C-terminal regions may includeany length of amino acids, which include the C-terminus, but do notinclude the N-terminus.

In certain embodiments, when referring to a polynucleotide, a region mayinclude a linear sequence of nucleic acids along the polynucleotide ormay include a three-dimensional area, secondary structure, or tertiarystructure. In certain embodiments, regions include terminal regions. Asused herein, the term “terminal region” refers to regions located at theends or termini of a given agent. When referring to polynucleotides,terminal regions may include 5′ and 3′ termini, 5′ termini refer to theend of a polynucleotide comprising a nucleic acid with a free phosphategroup, 3′ termini refer to the end of a polynucleotide comprising anucleic acid with a free hydroxyl group. 5′ and 3′ regions may there forinclude the 5′ and 3′ termini as well as surrounding nucleic acids. Incertain embodiments, 5′ and 3′ terminal regions include from about 9nucleic acids to about 90 nucleic acids, from about 15 nucleic acids toabout 120 nucleic acids, from about 30 nucleic acids to about 150nucleic acids, from about 60 nucleic acids to about 300 nucleic acidsand/or at least 300 nucleic acids. In certain embodiments, 5′ regionsmay include any length of nucleic acids that includes the 5′ terminusbut does not include the 3′ terminus. In certain embodiments, 3′ regionsmay include any length of nucleic acids, which include the 3′ terminus,but does not include the 5′ terminus.

RNA or RNA molecule: As used herein, the term “RNA” or “RNA molecule” or“ribonucleic acid molecule” refers to a polymer of ribonucleotides; theterm “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refersto a polymer of deoxyribonucleotides. DNA and RNA can be synthesizednaturally, e.g., by DNA replication and transcription of DNA,respectively; or be chemically synthesized. DNA and RNA can besingle-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded(e.g., double stranded, i.e., dsRNA and dsDNA, respectively). The term“mRNA” or “messenger RNA”, as used herein, refers to a single strandedRNA that encodes the amino acid sequence of one or more polypeptidechains.

RNA interfering or RNAi: As used herein, the term “RNA interfering” or“RNAi” refers to a sequence specific regulatory mechanism mediated byRNA molecules which results in the inhibition or interfering or“silencing” of the expression of a corresponding protein-coding gene.RNAi has been observed in many types of organisms, including plants,animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs(e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved fromfree dsRNA which direct the degradative mechanism to other similar RNAsequences. RNAi is controlled by the RNA-induced silencing complex(RISC) and is initiated by short/small dsRNA molecules in cellcytoplasm, where they interact with the catalytic RISC componentargonaute. The dsRNA molecules can be introduced into cells exogenously.Exogenous dsRNA initiates RNAi by activating the ribonuclease proteinDicer, which binds and cleaves dsRNAs to produce double-strandedfragments of 21-25 base pairs with a few unpaired overhang bases on eachend. These short double stranded fragments are called small interferingRNAs (siRNAs).

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Self-complementary viral particle: As used herein, a “self-complementaryviral particle” is a particle included of at least two components, aprotein capsid and a polynucleotide sequence encoding aself-complementary genome enclosed within the capsid.

Sense Strand: As used herein, the term “the sense strand” or “the secondstrand” or “the passenger strand” of a siRNA molecule refers to a strandthat is complementary to the antisense strand or first strand. Theantisense and sense strands of a siRNA molecule are hybridized to form aduplex structure. As used herein, a “siRNA duplex” includes a siRNAstrand having sufficient complementarity to a section of about 10-50nucleotides of the mRNA of the gene targeted for silencing and a siRNAstrand having sufficient complementarity to form a duplex with the othersiRNA strand.

Short interfering RNA or siRNA: As used herein, the terms “shortinterfering RNA,” “small interfering RNA” or “siRNA” refer to an RNAmolecule (or RNA analog) comprising between about 5-60 nucleotides (ornucleotide analogs) which is capable of directing or mediating RNAi. Incertain embodiments, a siRNA molecule includes between about 15-30nucleotides or nucleotide analogs, such as between about 16-25nucleotides (or nucleotide analogs), between about 18-23 nucleotides (ornucleotide analogs), between about 19-22 nucleotides (or nucleotideanalogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs),between about 19-25 nucleotides (or nucleotide analogs), and betweenabout 19-24 nucleotides (or nucleotide analogs). The term “short” siRNArefers to a siRNA comprising 5-23 nucleotides, such as 21 nucleotides(or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. Theterm “long” siRNA refers to a siRNA comprising 24-60 nucleotides, suchas about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides.Short siRNAs may, in some instances, include fewer than 19 nucleotides,e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, providedthat the shorter siRNA retains the ability to mediate RNAi. Likewise,long siRNAs may, in some instances, include more than 26 nucleotides,e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides,provided that the longer siRNA retains the ability to mediate RNAi ortranslational repression absent further processing, e.g., enzymaticprocessing, to a short siRNA, siRNAs can be single stranded RNAmolecules (ss-siRNAs) or double stranded RNA molecules (ds-siRNAs)comprising a sense strand and an antisense strand which hybridized toform a duplex structure called siRNA duplex.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event. In certainembodiments, a single unit dose is provided as a discrete dosage form(e.g., a tablet, capsule, patch, loaded syringe, vial, etc.).

Similarity. As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Spit dose: As used herein, a “split dose” is the division of single unitdose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and in certain embodiments, capable offormulation into an efficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the presentdisclosure may be administered, e.g., for experimental, diagnostic,prophylactic, and/or therapeutic purposes. Typical subjects includeanimals (e.g., mammals such as mice, rats, rabbits, non-human primates,and humans) and/or plants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In certainembodiments, an individual who is susceptible to a disease, disorder,and/or condition (for example, cancer) may be characterized by one ormore of the following: (1) a genetic mutation associated withdevelopment of the disease, disorder, and/or condition; (2) a geneticpolymorphism associated with development of the disease, disorder,and/or condition; (3) increased and/or decreased expression and/oractivity of a protein and/or nucleic acid associated with the disease,disorder, and/or condition; (4) habits and/or lifestyles associated withdevelopment of the disease, disorder, and/or condition; (5) a familyhistory of the disease, disorder, and/or condition; and (6) exposure toand/or infection with a microbe associated with development of thedisease, disorder, and/or condition. In certain embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionwill develop the disease, disorder, and/or condition. In certainembodiments, an individual who is susceptible to a disease, disorder,and/or condition will not develop the disease, disorder, and/orcondition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present disclosure may bechemical or enzymatic.

Targeting: As used herein, “targeting” means the process of design andselection of nucleic acid sequence that will hybridize to a targetnucleic acid and induce a desired effect.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, such as a mammal, a human, or a human patient.

Terminal region: As used herein, the term “terminal region” refers to aregion on the 5′ or 3′ end of a region of linked nucleosides or aminoacids (polynucleotide or polypeptide, respectively).

Terminally optimized: The term “terminally optimized” when referring tonucleic acids means the terminal regions of the nucleic acid areimproved in some way, e.g., codon optimized, over the native or wildtype terminal regions.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition. In certain embodiments, a therapeutically effectiveamount is provided in a single dose. In certain embodiments, atherapeutically effective amount is administered in a dosage regimencomprising a plurality of doses. Those skilled in the art willappreciate that in certain embodiments, a unit dosage form may beconsidered to include a therapeutically effective amount of a particularagent or entity if it includes an amount that is effective whenadministered as part of such a dosage regimen.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24-hour period. It may be administered as asingle unit dose.

Transfection: As used herein, the term “transfection” refers to methodsto introduce exogenous nucleic acids into a cell. Methods oftransfection include, but are not limited to, chemical methods, physicaltreatments and cationic lipids or mixtures.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Vector: As used herein, a “vector” is any molecule or moiety whichtransports, transduces or otherwise acts as a carrier of a heterologousmolecule. Vectors of the present disclosure may be producedrecombinantly and may be based on and/or may include adeno-associatedvirus (AAV) parent or reference sequence. Such parent or reference AAVsequences may serve as an original, second, third or subsequent sequencefor engineering vectors. In non-limiting examples, such parent orreference AAV sequences may include any one or more of the followingsequences: a polynucleotide sequence encoding a polypeptide ormulti-polypeptide, which sequence may be wild-type or modified fromwild-type and which sequence may encode full-length or partial sequenceof a protein, protein domain, or one or more subunits of a protein; apolynucleotide comprising a modulatory or regulatory nucleic acid whichsequence may be wild-type or modified from wild-type; and a transgenethat may or may not be modified from wild-type sequence. These AAVsequences may serve as either the “donor” sequence of one or more codons(at the nucleic acid level) or amino acids (at the polypeptide level) or“acceptor” sequences of one or more codons (at the nucleic acid level)or amino acids (at the polypeptide level).

Viral genome: As used herein, a “viral genome” or “vector genome” refersto the nucleic acid sequence(s) encapsulated in an AAV particle.

IV. Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments in accordance with the present disclosure described herein.The scope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The present disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thepresent disclosure includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the presentdisclosure, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the present disclosure(e.g., any antibiotic, therapeutic or active ingredient: any method ofproduction; any method of use; etc.) can be excluded from any one ormore claims, for any reason, whether or not related to the existence ofprior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the present disclosure in its broader aspects.

While the present disclosure has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the present disclosure.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1. Random Sequences with Altered G:C Content

To evaluate the effects of capsid incorporation ratios of GC content ofthe leader sequence of the 5′ UTR, a series of 5′ UTRs were designedfrom the parental gene leader sequences, Leader10 (AAV.rh10) and LeaderB(AAV.PRP.B). The leader sequences are given in Table 3, including thesize of the sequence ahead of the ATG start codon and the G:C content ofthe leader sequence in percentage.

TABLE 3 NAME LEADER SEQUENCE (5′-3′) Leader10-8TTAACACTTTAACCTTATAATACACATGCTGAGAATACTAATTATCGTAATGATGATAGTAT(GC25%99 bp-ATG) CCTATAGGAATTAATCTTATATAATGATTAGAAGCTT (SEQ ID NO: 59)Leader10-10 ACACACCGGTGGAGGCACGTCCTT (SEQ ID NO: 60) (GC62%24 bp-ATG)Leader10-16TGAGCACGGTAACCTTATAATACACCTGCCGAGAATACTAATTATCGTAATGATGATAGTAT(GC34%99 bp-ATG) CCTCTCGGAAGTAATCTTATATAATGTTTAGAAGCTT (SEQ ID NO: 61)Leader10-18TGAGCACGGTGACCTTAGAATACACCTGCCGAGTAAGCTCATTGTCGTAATGATGATAGTAT(GC50%99 bp-ATG) CCTCTCGGCATCAGTCTAGCCTGAGGCTTAGCCGCTT (SEQ ID NO: 62)Leader10-20TGAGCACGGTGACCTTAGAAGGCACCTGCCGAGTAGGCTCATTGTCGTCCTGATGATAGTAG(GC58%99 bp-ATG) CCTCTCGGCATGAGTCTCGCCTGCTGCGGAGCCGCTT (SEQ ID NO: 63)Leader10-2 Sequence of Leader10 parental serotype having CTG start codon(AcNPV-CTG) and baculovirus AcNPV Leader10-12Sequence of Leader10 parental serotype having leader sequence (L21-CTG)and CTG start codon Leader10-6Sequence of Leader10 parental serotype having CTG start codon(ACNPV-CTG-no and baculovirus AcNPV. Sequence not codon optimizedoptimization) Leader10-22Sequence of Leader10 parental serotype having the AAV2 leader(AAV2LS-wk3-ATG)sequence and Kozak sequence ATG-wk3 and ATG start codon. LeaderB-2Sequence of LeaderB parental serotype having the AAV2 leader(AAV2LS, codon sequence, CTG start codon and no ACG start codon for VP2.optimized, CTG, Sequence is codon optimized. no ACG for VP2

Each of the random leader sequences were inserted in the 5′ UTR of theLeader10 capsid gene ahead of the ATG start codon of VP11 to form theconstructs in Table 2. Western blot analysis was used to determine VP1,VP2 and VP3 expression. Results are shown in FIG. 3.

For Leader10-8 (GC25%99 bp-ATG), all capsid proteins were reduced. ForLeader10-10 (GC62%24 bp-ATG), the expression of VP1 was too strongrelative to VP3. For Leader10-16 (GC34%99 bp-ATG), all capsid proteinswere decreased but maintained a ratio close to 1:1:10 (VP1:VP2:VP3). ForLeader10-18 (GC50%99 bp-ATG), no results were obtained. For Leader10-20(GC58%99 bp-ATG), the overall yield was decreased and the levels werealtered such that VP2<VP1<<VP3.

Folding studies of the random leader sequences (SEQ ID No: 59-63) usingmFold software revealed the formation of a hairpin structure upstream(i.e., 5′) of the VP1 ATG start codon.

Example 2. Alteration of the Kozak Sequence

To explore the affect of the Kozak sequence strength on the capsidprotein expression ratio, various Kozak sequences were engineered intothe 5′ UTR of VP1 in two different AAV serotypes.

Three sequences were designed for the parental serotype. AAV2. Thesewere “ATG-wk1” (gggggatcctgttTAGatgGCTgccgacggttatctacccg; SEQ ID NO:64); “ATG-wk2” (gggggatectgttTAGatgTTGgccgacggttatctacccg; SEQ ID NO:65) and “ATG-wk3” (gggggatcctgttTITatgTTGgccgacggttatctacccg; SEQ ID NO:66). The start codons are identified in bold. The expressions of thecapsid proteins were determined by Western blot, with results shown inFIG. 4.

ATG-wk1 showed low expression of all capsid proteins, with minimal VP2expression and an unfavorable VP1:VP3 ratio of about 1:1. ATG-wk2 showedstrong over-expression of VP1, resulting in unfavorable VP1:VP3 ratiosabove 1:1. ATG-wk3 showed the best expression results, with VP1:VP2: VP3closest to 1:1:10.

The construct “Leader10-16 (AAV2WK3-ATG)” was then designed from ATG-wk3using Leader10 (AAV.rh10), having a sequence

(SEQ ID NO: 67 ACTCGACGAAGACTTGATCACCCGGGGGATCCTGTTTTTatgTTG;AAV2 leader sequence, ATG start codon in bold, Kozak sequence isunderlined). The Western blot for the construct is shown in FIG. 5. Thefigure shows both the full and empty capsids.

Example 3. Investigations of Hairpin (HP) Structure, Length andArrangement

Design characteristics which could lead to the ability to regulatecapsid protein expression, ratios and levels were evaluated, includingthe presence of mismatches, the size of the hairpin loop, the nature ofthe flanking sequences and the arrangement of certain features relativeto the start codon. The study involved the introduction of a hairpininto the 5′ UTR of a capsid gene, engineering the 5′ UTR size,alteration (e.g., weakening) the strength of the Kozak sequence, andde-optimization of the VP1 unique region.

The constructs designed and tested are given in Table 1. Theseconstructs were based on the parental AAV2 serotype. The ratio ofVP1:VP2:VP3 expression was determined, with results shown in FIG. 6A andFIG. 6B. FIG. 6A shows the expression levels of VP1, VP2 and VP3 for HP(hairpin) 6, 7, 8, and 9 at 3, 4, and 5 days post infection. FIG. 6Bshows the expression levels of VP1, VP2 and VP3 for HP (hairpin) 10, 12,13, 14, 15, and 16.

HP6 provided a VP1:VP3 ratio after 5 days of about 3.5:1, with no VP2.HP7 provided a VP1:VP3 ratio after 5 days of about 2:1, with no VP2. HP8provided a VP1:VP2:VP3 ratio after 5 days of about 2.6:1.3:10. HP9provided favorable results with a VP1:VP2:VP3 ratio after 4 days ofabout 1:0.5:10.

HP10 provided VP1:VP2:VP3 ratio of about 9.2:0.6:10. HP12 providedVP1:VP2:VP3 ratio of about 9.8:0.4:10. HP13 provided VP1:VP2:VP3 ratioof about 7.7:1:10. HP14 showed no expression. HP15 provided VP1:VP3ratio of about 2.5:1, with no VP2. HP16 provided VP1:VP2:VP3 ratio ofabout 17.7:0.3:10.

Example 4. Alternative Kozak Sequences

Studies involving the use of alternative Kozak sequences in 5′ UTRhairpins were performed to determine corresponding VP1, VP2 and VP3ratios.

Hairpin 9 from Table 1 was selected using an AAV2 parent serotype. TheKozak sequences tested are given in Table 4, as well as correspondingVP1:VP2:VP3 ratios.

TABLE 4 Concentration VP1:VP2:VP3 Name ng/ul Kozak Sequence ratioOriginal: TTT ATG GCT HP#9 1C-Att 92 ATT ATG GCT 33.7/0.8/10 2D-tAt 30TAT ATG GCT No Results 3C-ttA 25 TTA ATG GCT 1.8/0.1/10 4A-tAA 60TAA ATG GCT 4.2/0.3/10 5A-AtA 81 ATA ATG GCT 28.1/0.3/10

FIGS. 7A, 7B, 8A, 8B and 8C illustrate the VP protein expression levelsfor the different engineered polynucleotides of Table 4. These data showthat AAV2HP9-3C demonstrates a favorable expression level VP1 relativeto VP3, and that AAV2HP9-2D demonstrated a favorable VP1:VP2:VP3expression level.

We claim:
 1. An expression control sequence comprising a start codon andan engineered 5′ UTR polynucleotide; wherein the engineered 5′ UTRpolynucleotide encodes a hairpin structure which comprises, from 5′ to3′: (i) a 5′ flanking region which is 4-30 nucleotides in length; and(ii) a stem-loop structure comprising a stem region which is 4-30nucleotides in length, and a loop region which is 3′ of the stem regionand which is 4-15 nucleotides in length.
 2. The expression controlsequence of claim 1, wherein the start codon is ATG or ACG.
 3. Theexpression control sequence of claim 2, wherein the expression controlsequence comprises a Kozak sequence or modified Kozak sequence, andwherein the Kozak sequence or modified Kozak sequence comprises thestart codon.
 4. The expression control sequence of any one of claims1-3, wherein the 5′ flanking region is encoded by a nucleotide sequenceselected from atacgact, atac, and SEQ ID NO: 17-20.
 5. The expressioncontrol sequence of any one of claims 1-3, wherein the 5′ flankingregion is encoded by a nucleotide sequence having at least 60% identity,at least 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity or atleast 95% identity to a nucleotide sequence selected from atacgact,atac, and SEQ ID NO: 17-20.
 6. The expression control sequence of anyone of claims 1-5, wherein the stem region of the stem-loop structure isencoded by a nucleotide sequence selected from cggc, ctgcc and SEQ IDNO: 21-31.
 7. The expression control sequence of any one of claims 1-5,wherein the stem region of the stem-loop structure is encoded by anucleotide sequence having at least 60% identity, at least 65% identity,at least 70% identity, at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity or at least 95% identity to anucleotide sequence selected from cggc, ctgcc and SEQ ID NO: 21-31. 8.The expression control sequence of any one of claims 1-7, wherein theloop region of the stem-loop structure is encoded by a nucleotidesequence selected from tttatgget and atctaa.
 9. The expression controlsequence of any one of claims 1-7, wherein the loop region is encoded bya nucleotide sequence having at least 60% identity, at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity or at least 95%identity to a nucleotide sequence selected from tttatggct and atctaa.10. The expression control sequence of any one of claims 1-9, whereinthe stem-loop structure comprises a stem-complement region which is 3′of the stem region and which is 4-20 nucleotides in length.
 11. Theexpression control sequence of claim 10, wherein the stem-complementregion of the stem-loop structure is encoded by a nucleotide sequenceselected from ggcag and SEQ ID NO: 32-33.
 12. The expression controlsequence of claim 10, wherein the stem-complement region the stem-loopstructure is encoded by a nucleotide sequence having at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity or at least 95% identity to a nucleotide sequence selected fromggcag and SEQ ID NO: 32-33.
 13. The expression control sequence of anyone of claims 10-12, wherein the nucleotide sequence encoding thestem-complement region is 100% complementary to the nucleotide sequenceencoding the stem region.
 14. The expression control sequence of any oneof claims 10-12, wherein the nucleotide sequence encoding thestem-complement region and the nucleotide sequence encoding the stemregion are complimentary and include only one mismatch, only twomismatches, only three mismatches, only four mismatches or only fivemismatches.
 15. The expression control sequence of any one of claims1-14, wherein the hairpin structure comprises a 3′ flanking region whichis 3′ of the stem-loop structure and which is 4-30 nucleotides inlength.
 16. The expression control sequence of claim 15, wherein the 3′flanking region is encoded by a nucleotide sequence selected fromtttatgget, attatggct, tatatggct, ttaatggct, taaatggct, ataatggct, andSEQ ID NO: 34-35.
 17. The expression control sequence of claim 15,wherein the 3′ flanking region is encoded by a nucleotide sequencehaving at least 60% identity, at least 65% identity, at least 70%identity, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity or at least 95% identity to a nucleotidesequence selected from tttatggct, attatggct, tatatggct, ttaatggct,taaatggct, ataatggct, and SEQ ID NO: 34-35.
 18. The expression controlsequence of any one of claims 1-17, wherein the hairpin structurecomprises the start codon.
 19. The expression control sequence of anyone of claims 1-17, wherein the hairpin structure comprises the startcodon within the loop region of the stem-loop structure.
 20. Theexpression control sequence of any one of claims 10-14, wherein thehairpin structure comprises the start codon within the stem-complementregion of the stem-loop structure.
 21. The expression control sequenceof claim 20, wherein the distance between the 3′ end of the loop regionand the start codon is from 1-10 nucleotides.
 22. The expression controlsequence of any one of claims 15-17, wherein the hairpin structurecomprises the start codon within the 3′ flanking region.
 23. Theexpression control sequence of claim 22, wherein the distance betweenthe 3′ end of the stem-complement region and the start codon is from1-30 nucleotides.
 24. The expression control sequence of any one ofclaims 1-23, wherein the hairpin structure has a length from 10-100nucleotides, from 20-80 nucleotides, from 30-60 nucleotides, or from 35to 50 nucleotides.
 25. The expression control sequence of claim 1,wherein the hairpin structure is encoded by a nucleotide sequenceselected from SEQ ID NO: 1-16.
 26. The expression control sequence ofclaim 1, wherein the hairpin structure is encoded by the nucleotidesequence of SEQ ID NO:
 9. 27. The expression control sequence of claim1, wherein the hairpin structure is encoded by the nucleotide sequenceof SEQ ID NO:
 12. 28. The expression control sequence of any one ofclaims 1-24, wherein the expression control sequence comprises apromoter.
 29. The expression control sequence of claim 28, wherein thepromoter is a p10 promoter.
 30. The expression control sequence of claim28, wherein the promoter is a polH promoter.
 31. A viral expressionconstruct comprising a protein-coding nucleotide sequence, and theexpression control sequence of any one of claims 1-30; wherein theexpression control sequence is operably linked to the protein-codingnucleotide sequence.
 32. The viral expression construct of claim 31,wherein the protein-coding nucleotide sequence encodes a structural AAVcapsid protein or a non-structural AAV replication protein.
 33. Theviral expression construct of claim 31, wherein the protein-codingnucleotide sequence encodes a structural AAV capsid protein selectedfrom VP1, VP2, VP3, or a combination thereof.
 34. The viral expressionconstruct of claim 31, wherein the protein-coding nucleotide sequenceencodes a non-structural AAV replication protein selected from Rep78,Rep52, or a combination thereof.
 35. A method of producing proteinsencoded by a protein-coding nucleotide sequence, the method comprising:(i) introducing the viral expression construct of any one of claims31-32 into a viral production cell, and (ii) placing the viralproduction cell under conditions in which the cellular machinery of theviral production cell expresses the proteins encoded by theprotein-coding nucleotide sequence in the viral expression construct.36. The method of claim 35, wherein the protein-coding nucleotidesequence encodes VP1, VP2 and VP3 capsid proteins; and wherein theVP1:VP2:VP3 ratio of expressed proteins is 0.5-2.5:0.5-2.5:10.
 37. Themethod of claim 35, wherein the protein-coding nucleotide sequenceencodes VP1, VP2 and VP3 capsid proteins; and wherein the VP1:VP2:VP3ratio of expressed proteins is 1-2:1-2:10.
 38. The method of claim 35,wherein the protein-coding nucleotide sequence encodes Rep78 and Rep52replication proteins; and wherein the Rep78:Rep52 ratio of expressedproteins is between 1:1 and 1:20.
 39. The method of claim 35, whereinthe protein-coding nucleotide sequence encodes Rep78 and Rep52replication proteins; and wherein the Rep78:Rep52 ratio of expressedproteins is between 1:1 and 1:10.
 40. The method of claim 35, whereinthe protein-coding nucleotide sequence encodes Rep78 and Rep52replication proteins; and wherein the Rep78:Rep52 ratio of expressedproteins is between 1:1 and 1:5.